Response to Woodrow and Fanello

To the Editor—We thank Woodrow and Fanello [1] for their letter regarding our cross-sectional survey of pfhrp2-deleted Plasmodium falciparum in the Democratic Republic of Congo (DRC). We agree that polymerase chain reaction (PCR) methods are imperfect for proving the absence of a gene, especially in the setting of low DNA concentrations. However, we dispute their conclusion that the pfhrp2 deletion calls in our study are largely artifactual.

Woodrow and Fanello [1] point out that subjects included in the study initially had P. falciparum diagnosed by means of real-time PCR targeting the pfldh gene. They fail to note, however, that we used a relatively insensitive PCR assay with a limit of detection of approximately 100 parasites per microliter. In our hands, the typical limits of detection of the downstream pfhrp2 exon 1/2 and exon 2 assays are approximately 0.4 and 4 parasite genomes per microliter (10^–5 and 10^–4 ng/µL), respectively. Thus, the influence of stochastic PCR failure due to low DNA concentration is expected to be limited.

In addition, Woodrow and Fanello [1] highlight patterns of differential amplification of pfhrp2 exon 1/2 and exon 2 as evidence of borderline DNA concentrations. They also imply that published reports do not predict a high frequency of partial gene deletions. In fact, robust evidence supports the existence of partial pfhrp2 deletions due to chromosomal breakage in the subtelomere of chromosome 8 [2, 3]. Recent genomic analyses also confirm that the patterns and frequency of copy number variation in this region of the chromosome vary geographically, a finding supported by the heterogeneity of pfhrp2 deletion patterns reported in large surveys from different sites [4–8]. Therefore, deletion patterns that are distinct to a particular location are not unexpected. Nonetheless, we agree that the pattern and frequency of partial deletions we observed differ from findings in other large surveys and bear further study. Initial investigation suggests that assays for exon 1/2, but not exon 2, may amplify paralogous genes, producing a result that gives the appearance of a partial deletion when a complete deletion is actually present.

Furthermore, we agree that the testing algorithm can be improved. First, future studies should involve thorough analyses of both pfhrp2 and pfhrp3. Because we originally designed the study to focus on pfhrp2, we did not systematically evaluate the prevalence of pfhrp3 deletions in this survey, which we acknowledge could lead to an underestimation of their prevalence. Second, streamlined algorithms that assess both genes in parallel and incorporate simplified controls would permit high-throughput testing. We have since implemented these measures in ongoing studies of pfhrp2 and pfhrp3 deletions.

As with artemisinin resistance, the potential emergence of pfhrp2 and/or pfhrp3 (pfhrp2/3) gene deletions in Africa cannot be ignored. High rates of rapid diagnostic test (RDT) failure among symptomatic patients infected with pfhrp2/3-deleted P. falciparum in Eritrea are particularly concerning [9, 10]. In response, the World Health Organization has made it a priority to evaluate and address reports of false-negative RDT results [11].

We share and support the letter writers’ efforts to promote the thoughtful implementation of malaria diagnostic testing policies. Prematurely replacing HRP2-based RDTs with less sensitive, less heat-stable alternatives in the DRC would be akin to “throwing the baby out with the bathwater.” Our study findings indicate that these deletions are common among asymptomatic children in the DRC, and ongoing studies of their impact on symptomatic subjects of all ages are crucial for policy development.

Notes

Financial support.  This work was supported by the National Institute of Allergy and Infectious Diseases (grants 5T32AI007151 to J. B. P. and 5R01AI107949 to S. R. M.).

Potential conflicts of interest.  Both authors: No reported conflicts of interest. Both authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.