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Emergence of pfhrp2 and pfhrp3 Gene Deletions in Plasmodium falciparum and Diagnostic Challenges in Bangladesh

This study confirms that widespread *pfhrp2* and *pfhrp3* gene deletions in *Plasmodium falciparum* isolates from Bandarban, Bangladesh, are causing false-negative HRP2-based rapid diagnostic test results, thereby posing a significant threat to malaria elimination efforts and necessitating an urgent shift to alternative diagnostic strategies.

Original authors: Mohammad Shafiul Alam, Anamul Hasan, Md Mushfiqur Rahman, Amit Kumer Neogi, Md Mosiqure Rahaman, Shyamol Kumer Das, Afrida Tabassum Trina, Md. Muzahidul Islam, Nur-E Naznin Ferdous, Jane Cunningham, M

Published 2026-07-01

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CC BY 4.0

Original authors: Mohammad Shafiul Alam, Anamul Hasan, Md Mushfiqur Rahman, Amit Kumer Neogi, Md Mosiqure Rahaman, Shyamol Kumer Das, Afrida Tabassum Trina, Md. Muzahidul Islam, Nur-E Naznin Ferdous, Jane Cunningham, Md. Halimur Rashid

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

Imagine malaria as a sneaky intruder trying to break into a house. For years, the health workers in Bangladesh have used a specific "security camera" (a test called an RDT) to spot this intruder. This camera is designed to look for a very specific "uniform" that the malaria parasite wears: a protein called HRP2. If the camera sees the uniform, it sounds the alarm, and the patient gets treated.

However, this new study from Bandarban, Bangladesh, reveals that the intruder has started wearing a disguise.

The Great Disguise

The researchers found that in some areas, the malaria parasites have stopped wearing the HRP2 uniform entirely. In fact, they have deleted the genetic "blueprints" (genes) needed to make it. These are called pfhrp2 deletions. Even worse, most of these parasites (about 91% in this study) have also deleted the blueprints for a backup uniform called HRP3 (the pfhrp3 gene).

Think of it like a burglar who not only takes off their jacket but also throws away their ID card and any other identifying marks. When the security camera (the RDT) scans the blood, it sees nothing because the target is missing. The camera says, "All clear," but the intruder is actually still there.

The Detective Work

The study focused on 22 patients in Bandarban who were sick with fever.

The False Alarm: When these patients were tested with the standard "uniform-checking" camera (HRP2-based RDT), the result was negative. The camera said, "No malaria here."
The Real Truth: But when doctors looked at the blood under a powerful microscope (the old-school, trusted method), they saw the malaria parasites clearly.
The Confirmation: To be absolutely sure, the researchers used a high-tech DNA scanner (PCR). This confirmed that the patients definitely had Plasmodium falciparum (the deadliest type of malaria), but the parasites were missing the specific genes the camera was looking for.

What They Found

Total Deletion: Every single one of the 22 parasites was missing the main gene (pfhrp2).
Double Deletion: 20 out of 22 were missing both the main gene and the backup gene (pfhrp3).
The "Neighborhood" Effect: The researchers looked at the DNA surrounding these genes and found that the parasites didn't just delete the specific gene; they deleted a whole chunk of the neighborhood around it. It's like the burglar didn't just hide; they burned down the whole block to cover their tracks.

Why This Matters

The paper explains that this is a major problem because the "uniform-checking" cameras are the primary tool used in the field to diagnose malaria. If the camera can't see the parasite because the parasite has changed its disguise, sick people won't get treated. This allows the disease to spread silently and can lead to severe illness or death.

The study suggests that in this specific region of Bangladesh, the current "uniform-checking" tests are becoming unreliable for catching this specific type of malaria.

The Proposed Solution

The paper doesn't just point out the problem; it suggests a change in strategy based on what other countries have done:

Switch the Camera: Instead of looking for the missing "uniform" (HRP2), health workers should use a different type of camera that looks for a different part of the parasite's body (a protein called pLDH) that hasn't been deleted.
Don't Rely on Just One Tool: The study emphasizes that while the new cameras are needed, the old-school microscope is still a vital backup to catch cases the new cameras might miss.

The Bottom Line

This study is a warning siren. It shows that in Bandarban, the malaria parasite has evolved to hide from our most common detection tool. The researchers are calling for an urgent, wider survey to see how big this problem is across the country and to update the diagnostic rules so that no one is left undiagnosed and untreated. They want to ensure that the "security cameras" are updated to catch the new disguises before the disease spreads further.

Technical Summary: Emergence of pfhrp2 and pfhrp3 Gene Deletions in Plasmodium falciparum and Diagnostic Challenges in Bangladesh

Problem Statement
Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are a cornerstone of malaria elimination efforts globally. However, their efficacy is compromised in regions where Plasmodium falciparum parasites have developed deletions in the pfhrp2 and/or pfhrp3 genes, leading to false-negative results. While these deletions have been documented in South America, Africa, and parts of Asia, data regarding their prevalence and significance in Bangladesh remained limited prior to this study. The authors highlight a growing concern regarding "diagnostic discordance" in Bandarban, the district with the highest malaria burden in Bangladesh, where patients presenting with clinical malaria symptoms tested negative on routine HRP2-based RDTs but positive via microscopy and molecular assays.

Methodology
This descriptive laboratory study analyzed clinical samples collected from 2023 to 2025 in the hyper-endemic district of Bandarban. The study focused on 22 discordant cases: symptomatic individuals who tested negative on routine HRP2-based RDTs but were confirmed positive for P. falciparum via light microscopy and species-specific polymerase chain reaction (PCR).

The methodology involved a multi-step diagnostic and molecular workflow:

Sample Collection: Blood samples were obtained from patients in Alikadam, Rowangchhari, Lama, and Thanchi upazilas. Samples were collected via two pathways: direct collection of discordant cases and referrals from the National Malaria Elimination Programme (NMEP).
Diagnostic Confirmation: Initial screening used routine RDTs. All samples were re-tested in the laboratory using five different commercially available HRP2-based RDT platforms. Parasite density was quantified via Giemsa-stained thick and thin blood smears, with expert re-evaluation to ensure accuracy.
Molecular Characterization:
- Species Confirmation: SYBR Green-based real-time PCR confirmed P. falciparum mono-infections.
- Gene Deletion Screening: Conventional PCR was used to amplify exon-2 of pfhrp2 and pfhrp3, as well as selected flanking loci (MAL7P1_230, MAL7P1_228, MAL13P1-485, and MAL13P1-475) to distinguish between partial and complete deletions.
- Validation: Probe-based real-time PCR (qPCR) was employed to confirm the deletion status of pfhrp2 and pfhrp3, utilizing human $\beta$ -tubulin as an internal control for DNA integrity.

Key Results

Parasite Density: Microscopic examination revealed parasite densities ranging from 440 to 215,000 parasites/µL.
Gene Deletion Prevalence:
- Conventional PCR demonstrated pfhrp2 gene deletion in all 22 isolates (100%).
- 20 of the 22 isolates (90.9%) exhibited concurrent deletion of the pfhrp3 gene, indicating a predominance of double-deletion parasites.
- Probe-based real-time PCR confirmed these findings, detecting pfhrp2 deletion in all samples and pfhrp3 deletion in 20 samples.
Genomic Deletion Patterns: Analysis of flanking regions revealed that deletions extended beyond the target genes. The pfhrp2 locus showed a conserved loss pattern, specifically the loss of the upstream marker (MAL7P1_230) while retaining the downstream region. The pfhrp3 locus displayed greater variability, with the downstream marker deleted in all isolates but the upstream region retained in most.
RDT Performance: Despite high parasite densities in many cases, the HRP2-based RDTs consistently failed to detect the infection. In the two cases where pfhrp3 was retained, the RDTs remained negative, suggesting that HRP3 cross-reactivity was insufficient to generate a detectable signal at the observed parasitemia levels.

Significance and Claims
The authors claim that this study provides the first systematic confirmation of widespread pfhrp2 and pfhrp3 gene deletions in P. falciparum isolates from Bangladesh. The findings suggest that the current reliance on PfHRP2-based RDTs in the Bandarban region is leading to a "diagnostic blind spot," potentially allowing for ongoing transmission and threatening malaria elimination goals.

The paper posits that the high frequency of double deletions (~91%) indicates strong selective pressure, likely driven by the widespread use of HRP2-based RDTs introduced in Bangladesh in 2007. The authors emphasize that these deletions are not isolated events but represent a significant threat to diagnostic accuracy.

Limitations and Future Directions
The authors modestly acknowledge that the sample size (N=22) is small and not a random population sample, preventing an estimation of the overall prevalence of these deletions in the general population. Furthermore, the study was limited to the Bandarban district.

Consequently, the paper calls for:

Targeted Surveys: A broader survey across endemic regions to assess the scale and scope of the threat, specifically to determine if the WHO-recommended 5% deletion threshold is exceeded.
Diagnostic Strategy Adjustment: If deletion rates are confirmed to be high, the authors suggest transitioning to alternative diagnostic strategies, such as pLDH-based RDTs or combined antigen tests, to ensure accurate case detection.
Continued Surveillance: Ongoing monitoring of parasite genotypes is deemed essential to support effective malaria elimination efforts and prevent the silent transmission of gene-deleted parasites.

The study concludes that while microscopy and PCR remain critical reference standards, the emergence of these deletions necessitates a proactive shift in diagnostic policy to preserve recent progress in malaria control in Bangladesh.