Am. J. Trop. Med. Hyg., 97(4), 2017, pp.

1155–1158
doi:10.4269/ajtmh.16-0884
Copyright © 2017 by The American Society of Tropical Medicine and Hygiene

Case Report: A Case of Plasmodium falciparum hrp2 and hrp3 Gene Mutation in Bangladesh
Maisha Khair Nima,1 Thomas Hougard,1,2 Mohammad Enayet Hossain,1 Mohammad Golam Kibria,1 Abu Naser Mohon,1,3
Fatema Tuj Johora,1 Rajibur Rahman,1 Rashidul Haque,1 and Mohammad Shafiul Alam1*
1
International Centre for Diarrhoeal Disease Research Bangladesh (icddr,b), Dhaka 1212, Bangladesh; 2Department of Biochemistry,
Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota; 3Department of Microbiology and
Infectious Disease, Cumming School of Medicine, University of Calgary, Alberta T2N1N4, Canada

Abstract. Several species of Plasmodium are responsible for causing malaria in humans. Proper diagnoses are crucial
to case management, because severity and treatment varies between species. Diagnoses can be made using rapid
diagnostic tests (RDTs), which detect Plasmodium proteins. Plasmodium falciparum causes the most virulent cases of
malaria, and P. falciparum histidine-rich protein 2 (PfHRP2) is a common target of falciparum malaria RDTs. Here we report
a case in which a falciparum malaria patient in Bangladesh tested negative on PfHRP2-based RDTs. The negative results
can be attributed to a deletion of part of the pfhrp2 gene and frameshift mutations in both pfhrp2 and pfhrp3 gene. This
finding may have implications for malaria diagnostics and case management in Bangladesh and other regions of South
Asia.

More than 90% of symptomatic malaria cases in Bangla- a microscope. Falciparum malaria was diagnosed and, as per
desh are caused by Plasmodium falciparum.1 Species- national guideline, the patient was treated with artemisinin-
specific diagnoses are crucial to proper case management based combination therapy (ACT) for 3 days. A written in-
because falciparum malaria cases require different treatments formed consent was obtained from the patient, and the
and can rapidly lead to severe complications and death within original study was approved by the institutional ethics review
days. Rapid diagnostic tests (RDTs) are extensively used for committee of the International Center for Diarrheal Disease
fast and proper diagnosis, and they are accessible even in Research, Bangladesh (icddr,b). Venous blood was obtained
areas without electricity or trained microscopists.2 from the patient in ethylenediaminetetraacetic acid (EDTA)
Commonly used RDTs that are capable of specifically tubes and sent to icddr,b for further testing.
detecting P. falciparum target either P. falciparum histidine- Presence of P. falciparum was confirmed via reexamination
rich protein 2 (PfHRP2) or P. falciparum lactate de- by two expert microscopists, and a parasitemia of 5,120
hydrogenase (PfLDH);3 PfHRP2 has several repeats of the parasites/mL was counted in a thin blood film, corresponding
antibody-binding epitopes,4 which makes it ideal for immu- to 0.1024% of erythrocytes infected. The blood sample was
nodetection by RDT.5 PfHRP3 can also be detected by tested again, using three brands of RDTs: InTec® Pan/Pf
PfHRP2 RDTs because of the sequence homology of pfhrp2 Combo and Pf/Pv Combo (InTec Products Inc., Xiamen,
and pfhrp3 gene although the expression is much lower than China), SD Bioline Pf/Pv Combo (Standard Diagnostics, Yongin,
PfHRP2.5,6 PfHRP2 RDTs also have limitations. The pfhrp2 Republic of Korea), and Falcivax Pv/Pf Combo (Falcivax; Zephyr
gene has greater variability than the pfldh gene,7 and pfhrp2 Biomedicals, India). The sample tested negative for PfHRP2
polymorphism can affect its detection by RDTs.5 In addition, on each RDT and positive for pLDH, shown by the “PAN” line
strains with partial or total pfhrp2 deletions have been reported on the InTec test. Diagnostic polymerase chain reaction
in South America, Africa, and India.3,8–10 A recent study in (PCR) reconfirmed P. falciparum infection following a pro-
India reported 2.4% and 1.8% prevalence of pfhrp2 and tocol described previously.12 In addition, msp2 marker (3D7
pfhrp3 gene deletion, respectively; including an area in Tripura of central domain) region was amplified to confirm the validity
state, which shares its border with our study area.11 Here we of the DNA preparation.13,14
report a case in which a falciparum malaria blood sample Given the high parasitemia of the sample (KHC 225) and
tested negative on a PfHRP2-based RDT in Bangladesh. A PCR-confirmed diagnosis, variation or deletion in the hrp2 and
molecular basis for the negative result is provided, and its hrp3 gene was the suspected cause for negative RDT results.
implications are discussed. The sample was subjected to PCR targeting the hrp2 and hrp3
In August 2013, a blood sample was obtained from a gene. Two pairs of primers targeting the untranslated region
24-year-old male with high fever, chills, nausea, and headache (UTR), exon 1 and intronic region (PCR reaction 1), and exon 2
at the Kamalganj Upazilla Health Complex in Sylhet, Bangla- region (PCR reaction 2) of pfhrp2 were derived from a previous
desh, located at 91°509600 E longitude and 24°219360 N lati- study.15 The other two sets of primers targeting the same sites
tude. The patient had no records of traveling abroad. The (PCR reaction 3 and 4) of pfhrp3 gene were derived from other
Falcivax Pv/Pf Combo RDT (Zephyr Biomedicals, Verna, Goa, studies.16,17 The reverse primer for PCR reaction 4 is the re-
India) was used, and the sample tested negative for both verse compliment of the forward primer for PCR reaction 3.
PfHRP2 and Plasmodium vivax lactate dehydrogenase PCR cycle conditions were thus modified; for 35 cycles, the
(PvLDH). However, because the patient’s symptoms reflec- annealing temperature for PCR reactions 1 and 3 was set to
ted falciparum malaria, a thin blood film was examined under 55°C for 35 seconds and extension temperature was set to
68°C for 40 seconds followed by final extension at 68°C for
10 minutes. For PCR reaction 2 and 4, the conditions and cy-
* Address correspondence to Mohammad Shafiul Alam, International
Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68
cles were the same, except the annealing stage was modified to
Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangla- 30 seconds and extension stage was 1 minute and 20 seconds.
desh. E-mail: shafi[email protected] Both reactions included DNA extracted from a P. falciparum

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culture as a positive control (MRA-156, MR4, ATCC, Mana- In pfhrp2 exon 2, several point mutations and insertions
ssas, VA). Water was used as a common negative control were found, as well as a frameshift deletion very early in exon
along with genomic DNA of strain D10 and HB3 (MRA-201G 2. The aligned sequence of KHC 225 hrp2 shares 93% identity
and MRA-155G, MR4, ATCC, Manassas, VA) as negative with PF3D7_0831800. In KHC 225 pfhrp3, the region spanning
control for hrp2 and hrp3 gene amplification, respectively. UTR and exon 1 was conserved with some single-nucleotide
Amplification products were visualized by gel electrophoresis. polymorphisms (SNPs) in the intronic region. The exon 2
In the hrp2 gene amplification cycle, PCR reaction 1 yielded portion had various SNPs along with a 3 bp insertion and a
no visible amplification product for the experimental sample. frameshift deletion. KHC 225 hrp3 and PF3D7_1372200 share
PCR reaction 2 provided the expected DNA fragment approx- 81% identity. PCR and sequencing was performed at least
imately 1,021 bp long. For the hrp3 amplification, PCR reac- three times for each portion of both the genes to confirm the
tions 3 and 4 yielded fragments of approximately 460 and mutations. The partial exon 2 sequences of KHC 225 hrp2
483 bp. The amplified products were sequenced using and hrp3 reported in this article has been deposited in the
ABI3500 Genetic Analyzer (Life Technologies, Foster City, CA,) GenBank database (accession number KX388531 and
after purification by ExoSAP-IT (Affymetrix). ClustalW was used MF176231).
to align the sequences with that of P. falciparum strain 3D7 Low parasitemia is one possible explanation for false neg-
(PF3D7_0831800 and PF3D7_1372200, PlasmoDB Release 28). atives using RDTs, but this was ruled out as the parasitemia of

FIGURE 1. (A) HRP2 alignment of KHC 225 with PF3D7_0831800 (3D7). Major changes in the HRP2 amino acid sequence of KHC 225 include
insertion of eight amino acids after the 83 position, deletion of positions 113–115, insertion of six amino acids after the 219th and mismatches in
several positions. (B) HRP3 alignment of KHC 225 with PF3D7_1372200 (3D7). Several major mismatches between KHC 225 HRP3 amino acid
sequence and PF3D7_1372200 were found from position 96 to 152 and 221 to 231. This figure appears in color at www.ajtmh.org.
 P. FALCIPARUM HRP2 AND HRP3 GENE MUTATION IN BANGLADESH 1157

5,120 parasites/mL, which is approximately 50 times than that Rashidul Haque, and Mohammad Shafiul Alam, International Centre
normally required for RDTs.5 Interestingly, PfHRP3 can also for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka 1212,
Bangladesh, E-mails: [email protected], houga006@umn.
bind to PfHRP2-based RDTs. It has been found, however, that edu, [email protected], [email protected], rajibur.
when PfHRP2 is missing, PfHRP3 expression is reduced.6 [email protected], [email protected], and shafi[email protected].
The lack of amplification in PCR reaction 1 suggests a de- Thomas Hougard, Department of Biochemistry, Molecular Biology
letion upstream of the forward primer for reaction 2, which is and Biophysics, University of Minnesota, Minneapolis, MN, E-mail:
[email protected]. Abu Naser Mohon, Department of Microbiology
consistent with other strains found to have partial pfhrp2 de- and Infectious Disease, Cumming School of Medicine, University of
letions.9 Chromosome breakage in this unstable subtelomeric Calgary, Alberta T2N1N4, Canada, E-mail: [email protected].
region is often accompanied by addition of new telomeric se-
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