First evidence of the deletion in the Pfhrp2 and Pfhrp3 genes in P. falciparum from Equatorial Guinea

Background WHO recommends RDTs as a good alternative malaria-diagnosis method in remote parts of sub-Saharan Africa. The majority of commercial RDTs currently available detect the P. falciparum protein histidine-rich protein 2 (PfHRP2). There have also been recent reports of Pfhrp2 deletions being found in parasites collected from several African countries. WHO has concluded that the lacking the Pfhrp2 gene must be monitored in Africa. The purpose of the study was to analyze why the samples that were positive by PCR were negative by RDTs; and, therefore, to determine whether there have been deletions in the Pfhrp2 and/or Pfhrp3 genes. Methods Malaria NM-PCR was carried out to all the samples collected in the field. A group of 128 samples was positive by PCR but negatives by RDT, these samples were classified as RDT false-negatives. It was carried out a PCR for exon2 of Pfhrp2 and Pfhrp3 genes to detect the presence or absence of these two genes. Frequencies with 95% confidence intervals (CIs) were used for prevalence estimates. Associations were assessed by the chi-square test or Fisher´s exact test. The level of significance was set at p ≤ 0.05. Statistical analyses were performed using the software package SPSSv.15.0. Results After the PCR, 81 samples were identified (4.7%, 95%CI: 3.8-5.8) which had deletion in both genes, Pfhrp2 and Pfhrp3 . Overall, however, 11 samples (0.6%, 95%CI: 0.36-1.14) had deletion only in Pfhrp2 but not in Pfhrp3 , and 15 (0.9%, 95%CI: 0.6-1.5) presented with deletion only in Pfhrp3 but not in Pfhrp2 . Considering the Pfhrp2 gene separately, within the total of 1,724 samples, 92 (5.3%, 95%CI: 4.37-6.5) had evidence of deletion. study This test enables differential diagnosis between P. falciparum (Pf), Plasmodium malariae (Pm), P. vivax (Pv), and P. ovale (Po) in human whole-blood samples . It detects HRP2-specific proteins for Pf, and pLDH-specific proteins for Pf, Pv, Pm, and Po. The test has a sensitivity of 99.7% for Pf and 95.5% for non-Pf parasites with the microscopic detail of a large droplet, and a specificity of 99.5%. The cut-off level was 1 to 50 parasites/ml of blood for HRP2 and 51 to 100 parasites/ml of blood for pLDH. To perform the malaria test, 5 ml of whole blood is collected with the provided capillary pipette and transferred to the sample well. Four drops of the assay diluent are then added to the diluent well, in accordance with the manufacturer’s protocol. The results are read after 15-20 min, only tests containing the control band are considered valid. Participants whose RDTs produced positive results were immediately offered treatment as set out by the EG national guidelines [22].

The RDT used in situ was the NADAL ® Malaria 4 species test (Test cassette) (Nal von Minden, Moers, Germany). This test enables differential diagnosis between P. falciparum (Pf), Plasmodium malariae (Pm), P. vivax (Pv), and P. ovale (Po) in human whole-blood samples. It detects HRP2-specific proteins for Pf, and pLDH-specific proteins for Pf, Pv, Pm, and Po. The test has a sensitivity of 99.7% for Pf and 95.5% for non-Pf parasites with the microscopic detail of a large droplet, and a specificity of 99.5%.
The cut-off level was 1 to 50 parasites/ml of blood for HRP2 and 51 to 100 parasites/ml of blood for pLDH. To perform the malaria test, 5 ml of whole blood is collected with the provided capillary pipette and transferred to the sample well. Four drops of the assay diluent are then added to the diluent well, in accordance with the manufacturer's protocol. The results are read after 15-20 min, only tests containing the control band are considered valid. Participants whose RDTs produced positive results were immediately offered treatment as set out by the EG national guidelines [22].

DNA extraction and molecular analysis
The DNA was extracted from the filter paper samples using commercial kits (Speedtools tissue DNA Extraction Kit, Biotools, Spain). It was used a 5 mm diameter punch that contained 10ml of blood.

1.
NM-PCR for the diagnosis of malaria: the target selected in this NM-PCR is the gene encoding the 18S small subunit ribosome RNA (ssrDNA) and includes an internal amplification control to avoid false negatives (18S human rRNA) [23][24][25]. This was carried out on all the samples collected in the field, including both positives and negatives by microscopy and by RDTs irrespective of the result. After the NM-PCR tests had been completed, a group of 128 samples was identified for further study; these samples were positive using PCR and by microscopy, but negative using RDTs.

Statistical analysis
Frequencies with 95% confidence intervals (CIs) were used for prevalence estimates. Associations were assessed by the chi-square test or Fisher´s exact test. The level of significance was set at p ≤ 0.05. Statistical analyses were performed using the software package SPSSv.15.0.

Results
A total of 1,724 blood samples were diagnosed by microscopy, NM-PCR and RDT [21]. The Plasmodium sp. samples marked as negative by both microscopy and RDT were tested by NM-PCR as a quality control of the diagnoses. In this group of negative-by-RDT samples (n=963), 128 (7.4%) were identified as false negatives by PCR and were diagnosed as: 122 P. falciparum, 1 P. falciparum/P. vivax, 1 P. malariae, 1 P. vivax and 1 P. ovale. Figure 2 shows how the 1,724 samples were processed.
These 128 negative by RDT samples could have been due to a number of possible causes: deletion in the Pfhrp2 or Pfhrp3 genes, a technicians' misinterpretation, or that there is a parasitemia in the sample that is lower than the minimum detection threshold for RDTs..
The DNA from the 128 false negatives by RDT were amplified correctly by the nested-PCR for the following genes of P. falciparum ( Figure 3): Pfdhfr (108/164 and 51/59, product sizes 254bp and 113bp, respectively), Pfdhps (400 and 500, product sizes 148bp and 201bp, respectively), Pfmdr1 (86 and 1246, product sizes 203bp and 295bp, respectively) and Pfcrt (76, product size 145bp). This indicates that the DNA has been extracted correctly, that it has no inhibition factors for PCR and that it has sufficient concentration to be used successfully in the PCR of exon2 of Pfhdrp2 and Pfhrp3 genes. All the DNA samples were studied for the deletion of Pfhrp2 and Pfhrp3 using PCR (PCR for exon2 of the pfhrp2 and Pfhrp3 genes) the sizes of the expected fragments, if amplification did occur, were +/-814bp for Pfhrp2 and +/-719bp for Pfhrp3; this determined the presence or absence of these genes in the samples, Figure 4 shows the result of the PCR tests. The decision was made to perform the PCR for exon2 of Pfhrp2 / 3 in all samples that were negative for RDT and positive for NM-PCR, although in some samples were diagnosed as non-falciparum. In the non-falciparum samples amplification fragment for Pfhrp2 / 3 was not to be obtained. In this way it was also test the specificity of the PCR that is only valid for Pf, not giving false positives, that is, the non-falciparum samples acted as negative controls.
After the carrying out the PCR on the 128 RDT false-negative samples, the 5 non-falciparum (1 Pm, 3 Po and 1 Pv) samples were, as expected, negative in the Pfhrp2/Pfhrp3-PCR. The mix sample (Pf/Pv) were negative in the PCR for these two genes, therefore, was detected deletion in these genes for and if not possible, confirm the result of the RDT by another technique such as microcopy. This study identified 5.3% Pfhrp2 deletion in the regional sample; moreover, this shows that it is now necessary to monitor the deletion of this gene across the whole country in order to obtain a complete picture of the deletions occurring with these genes. It is important to remember that this study was carried out in a district of Equatorial Guinea's continental region; therefore, it is evident that the study needs to be extended to cover the country in its entirety.
Deletions in Pfhrp3 were also detected in the study, although the RDT used in EG and for this study was not designed to detect Pfhrp3 proteins. In most settings, genetic mutations like Pfhrp2/Pfhrp3 deletion in parasites are unlikely to be the main cause of RDT false-negatives unlike in this study and more studies are required to establish the true prevalence of these mutations in EG. In fact, there were some samples that were RDT false-negatives which were found to positive using NM-PCR, but without any detectable deletion in the Phrp2 and Pfhrp3 genes. Therefore, these results might be due to problems with the RDT used itself, or as result of operator errors when carrying out the tests and/or the interpretation RDT results; all of these could result in false-negatives [34].
Attributing false-negatives to Pfhrp2/Pfhrp3 deletion has significant implications for public health policy. Once it has been established that the threshold has been passed, alternative RDTs will have to be procured and case management decisions will have to be revised, with re-training in the use of the new RDTs. Therefore, investigation into such deletions must be carried out systematically and accurately [7]. If Pfhrp2 deletions are found to be prevalent among symptomatic individuals (the lower 95% confidence interval is still above 5%) as is the case, for example, in Eritrea and several countries in South America (Brazil, Colombia, Peru) national malaria control programs will have to switch to RDTs that do not exclusively rely on PfHRP2 to detect P. falciparum. A 5% threshold was selected by the WHO because it is somewhere around this point that the proportion of cases missed by PfHRP2 RDTs due to non-hrp2 expression is likely to be greater than that which would be missed by using less-sensitive pLDH-based RDTs. A recommendation to switch can be further informed by mathematical modelling which shows whether parasites lacking PfHRP2 genes will spread under PfHRP2-only RDT pressure; policy makers may also decide to switch because of the complexity of procuring multiple RDTs and training staff in their use. In general, any change should be applied nationwide, although roll-out might be prioritized on the basis of PfHRP2-deletion prevalence in a given region [35]. Where microscopy is available, services should be strengthened to ensure that parasitological confirmation continues during the transition to new RDTs, and in order to investigate new suspected PfHRP2/PfHRP3 deleted parasite foci.
Excessive use of Pfhrp-based RDTs might enhance the selection of P. falciparum isolates with Pfhrp2 deletion, especially in endemic areas where Pfhrp2 deletion is present, as the case in Equatorial Guinea. Previous reports have also shown that Pfhrp3 deletion can be an early warning sign for Pfhrp2 deletion. Thus, it is important to monitor the presence of parasites with Pfhrp2 deletions to avoid RDT false-negatives as well as Pfhrp3 deletions to act as an early warning which offers public health bodies an opportunity to step up monitoring efforts and consider longer-term contingency plans [8,9].

Conclusion
The RDTs used in this study detected the majority of P. falciparum infections as well as those from other species. Regarding with the deletion of the genes, it would be strongly recommendable to implement an active surveillance program in order to detect any increases in Pfhrp2 and Pfhrp3 deletion frequencies. Although there are false negatives due to causes other than deletion of these genes, a surveillance program is critical due to the level of frequencies of the deletion detected in the study. This surveillance could be implemented through different regions and different seasonal profiles, needed to determine the full extent of Pfhrp2 and Pfhrp3 deletion.
To be able to control malaria, it is essential to have good diagnostic tools on the front line. To this end, the present study provides the first evidence of deletion in the Pfhrp2 and Pfhrp3 genes in P.     Results of the Nested PCR for Pfdhfr, Pfdhps, Pfmdr1 and Pfcrt genes: amplification appears in all cases, therefore, it indicates that the DNA was well extracted and works correctly in PCR. These PCRs are used as a control, all samples amplified perfectly so when no amplification appears in pfhrp2 / 3 indicates for sure that there is deletion, it is not a problem with the DNA. lines 1 / 2 (+/-719bp for Pfhrp3). When the fragments do not appear indicate that deletion exists. ; fragments are sequenced to confirm that they correspond to the pfhrp2 / 3