The WHO recently recommended adopting universal testing to confirm the presence of malaria parasites prior to the use of ACT, and in the cases where microscopic examination cannot be performed, the RDT would be the best alternative for confirmation. In 2006, the Senegalese National Malaria Control Programme recommended ACT as the first-line treatment for uncomplicated malaria and, in 2007, mandated testing for all suspected cases of malaria with P. falciparum using the HRP2-based RDT. PfHRP2-based RDTs are now commonly used with high adherence in Senegal
[17–19]. These RDTs can provide a quick and accurate diagnosis, thereby leading to timely and appropriate malaria treatment and a reduction in the severity and economic burden of the disease.
In the present work, 5.9% of the total (n = 7) P. falciparum isolates and 10.7% of the isolates with parasitaemia ≤0.005% (≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT. Moreover, of the isolates detected as P. falciparum by PfHRP2, 7.1% were not detected by the pan-malaria-specific pLDH. Five P. falciparum isolates (4.2%) were misdiagnosed as P. vivax parasites by the P. vivax LDH.
Three possible factors can affect the sensitivity of the PfHRP2-based RDT: parasite density, pfhrp2 deletion and pfhrp2 polymorphisms. The parasite density can not explain the failure of the detection by the PfHRP2 Palutop+4® RDT. There was no significant difference in term of sample numbers (p = 0.246, Chi-square test) or in term of parasitaemia between detected (mean = 0.45%; standard deviation = 1.16%) and undetected samples by HRP2 (mean = 0.22%; standard deviation = 0.26%) (p = 0.595, Wilcoxon rank sum test). In addition, the non-detection by the pan-malaria-specific pLDH was not associated with parasite density. There was no significantly difference in term of parasitaemia between the two groups (p = 0.144, Wilcoxon rank sum test). For the five P. falciparum isolates (4.2%) misdiagnosed as P. vivax parasites by the P. vivax LDH, all of these showed parasitaemia ≥0.005% (≥250 parasites/μl).
Another possible factor affecting the sensitivity of the PfHRP2-based RDT is the failure of the parasite to express the antigen, either due to genetic deletions, frame shift mutations or alterations in protein expression. The deletions of pfhrp2 and pfhrp3 were reported as the cause of false-negative diagnoses in populations from Peru
[8, 9], Mali
 and in a clinical case from Brazil
. In addition, the levels of pfhrp2 transcription and PfHRP2 protein expression varied between P. falciparum strains, and this may impact the detection sensitivity of the PfHRP2-based RDT
. The three samples with deletion of pfhrp2 (2.4%) were not detected by the PfHRP2-based Palutop+4®. The deletion of pfhrp2 is one of the factors of false-negative diagnoses using PfHRP2-based RDT. Of the 16 samples with deletion of pfhrp3 (12.8%), six were not detected by the PfHRP2-based Palutop+4®. The frequency of pfhrp2 deletion observed in Senegal is comparable to those estimated in Africa, like in Mali (2%)
 or in India (4.2%)
 and lower than those estimated in South America like in Peru (25.7 and 41%)
[8, 9]. As previously described in Peru
, the proportion of parasites lacking pfhrp3 is higher than those lacking pfhrp2 in samples collected in Dakar. This suggests that parasites lacking pfhrp3 may have been present in Senegal longer than those lacking pfhrp2.
Polymorphisms in the pfhrp2 gene that can affect the sensitivity of PfHRP2-based RDTs have been detected in the Asia-Pacific region
[4, 5], India
 and in a clinical case in Uganda
. Prior to this report, no data were yet available on the sequence variation of HRP2 from P. falciparum parasites from Senegal. Consistent with previous reports
[5–7], the PfHRP2 was highly diverse in parasite isolates from Dakar. Of the 122 PfHRP2 sequences, 120 unique sequences were identified. In previous works, all pfhrp2 sequences have begun with a type 1 repeat (AHHAHHVAD)
[5–7]. However, three of the Senegalese isolate sequences in this study did not begin with a type 1 repeat. The type 11 repeat (AHN) was not detected in P. falciparum parasites from Dakar. This is consistent with isolates from Africa
, except for one isolate from Cameroon
 and one from northern Madagascar
. However, the Senegalese PfHRP2 sequences had certain characteristics: only one isolate possessed a type 12 repeat (AHHAAAHHEAATH), while the PfHRP2 from all the previously described isolates from Africa, the Pacific, South America or Asia contained one type 12 repeat
[3, 5]. The type 9 repeat has not been described in Africa
[3–5]. Interestingly, two isolates from Dakar showed a type 9 repeat. Types 13 and 14 have rarely been observed in Africa
[3–5], yet these two types of repeats were found in nine (7.4%) and five (4.1%) isolates from Dakar, respectively.
Finally, using the Baker’s regression model, it was shown that at least 7.4% of P. falciparum isolates in Dakar (group C) were likely to be undetected by PfHRP2-based RDT at a parasite density of ≤250 parasites/μl. In Madagascar, 9% of the isolates at a parasite density of ≤250 parasites/μl were predicted to be undetected by PfHRP2-based RDT. The number of isolates predicted to be non-sensitive to the PfHRP2-based RDT increased significantly between 2009 and 2011 (0% in 2009, 5% in 2010 and 16.3% in 2011; P = 0.0046). One hypothesis to explain this increase is the selection of parasites which contain less than 43 repeats (type 2 × type 7 repeats). PfHRP2-based RDTs are now commonly used with high adherence in Senegal
[17–19]. Patients with negative PfHRP2-based RDT are not treated for malaria. If the parasites are undetected by the PfHRP2-based RDT, the delay of diagnose before treatment might permit an increase in the time required for development of sexual stages (gametocytes) and in their transmission to mosquitoes during a blood meal compared to parasites treated quickly. The transmission of parasites undetected by the PfHRP2-based RDT might be more important, leading to faster dissemination of these genotypes. The frequency of these genotypes could increase in coming years. This evolution should continue to be monitored in Senegal.
In the Dakar samples with low parasite density, the predicted prevalence was slightly lower than the evaluated prevalence (7.4% versus 10.7%). The predictive regression model developed by Baker cannot explain all of the PfHRP2 non-detection in Senegal. Of the four samples not detected by the PfHRP2-based Palutop+4® but with PfHRP2, only one (183/3) was classified in group C (non-sensitive) according to the Baker’s regression model. In addition, of the nine samples expected to be not detected by PfHRP2-based RDT, the sample 183/3 was the only one which was not detected by the PfHRP2-based Palutop+4®. However, this sample was the only one with parasitaemia ≤0.005% (≤250 parasites/μl).
It has been reported that, due to PfHRP3 and PfHRP2 structural homology, PfHRP3 can cross-react with HRP2-coated antibodies in the RDT. PfHRP3 has also contributed to the detection of P. falciparum malaria infections
. In this study, the genetic diversity of pfhrp3 (64 different sequences) was less than that of pfhrp2 (120 different sequences). All of the previously described repeat types, other than type 2, were present (99% to 100%). The type 2 repeat has never been described in HRP3 in Africa
[3, 4]. The Senegalese HRP3 sequences had characteristics in common with those of other African isolates
[3, 4] and were different from Indian isolates in that they did not contain type 1 repeats
. The most common repeat types between PfHRP2 and PfHRP3 in Africa are types 1 and 7. These shared repeats, particularly type 7, which is the only type described both in Africa and India, are likely the basis for the observed cross-reactivity between PfHRP3- and PfHRP2-specific monoclonal antibodies
[21, 22]. In addition, the type 7 repeat is one of the two selected types in the Baker’s regression model to predict non-detection of parasites by PfHRP2 at a density of ≤250 parasites/μl.