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Table of Contents
Year : 2021  |  Volume : 58  |  Issue : 3  |  Page : 273-280

Prevalence of Pfhrp2/3 gene deletions among false negative rapid antigen test results in central India

1 ICMR-National Institute of Malaria Research, Sector-8, Dwarka, New Delhi; Department of Biotechnology, Kumaun University, Bhimtal, Uttarakhand, India
2 ICMR-National Institute of Malaria Research, Sector-8, Dwarka, New Delhi, India
3 Department of Biotechnology, Kumaun University, Bhimtal, Uttarakhand, India

Date of Submission01-Mar-2020
Date of Acceptance21-Aug-2020
Date of Web Publication15-Feb-2022

Correspondence Address:
Dr. Anupkumar R Anvikar
Scientist F, ICMR-National Institute of Malaria Research, Sector-8, Dwarka, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.328815

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Background &objectives: The diagnosis of Plasmodium falciparum malaria is widely dependent on the P. falciparum histidine rich protein 2 (PfHRP2) antigens based rapid diagnostic tests. There are few possible factors like Pfhrp2 polymorphism, Pfhrp2 deletion and density of malaria parasite which can affect the sensitivity of the Pf-HRP2-based RDT. The primary objective of the investigation was to check whether the Pfhrp2 gene deletion is the primary cause of RDT false negative cases.
Methods: Febrile patients from three districts of Chhattisgarh, India were screened for malaria during 2016–2017 by microscopy and RDT. All microscopy P. falciparum positive samples were validated by PCR. Microscopy positive and RDT negative samples were analyzed for the presence of Exon 2, across Exon 1-2, upstream and downstream of both the Pfhrp2 and Pfhrp3 genes fragment by PCR.
Results: Out of 203 screened samples, 85 were detected positive for P. falciparum malaria based on microscopy and PCR. Among these 85 P. falciparum positive samples, 4 samples were observed Pf-HRP2 RDT negative. Although, it signified that the RDTs used were reliable with sensitivity of 95.3% (81/85). 3/4 PfHRP2-RDT negative samples of the P. falciparum isolates exhibited complete deletion of Pfhrp2 and Pfhrp3 genes and one sample was found RDT false negative due to high parasite density.
Interpretation & conclusion: Pfhrp2 and Pfhrp3 deletions that result in false negative RDTs were uncommon in our setting. The continued monitoring of RDTS which results in false negative tests due to Pfhrp2/3 gene deletion is the need of the hour for an effective malaria elimination strategy.

Keywords: Malaria; P. falciparum; RDTs; Pfhrp2/Pfhrp3; Gene deletions

How to cite this article:
Kumari S, Ahmed MZ, Sharma S, Pande V, Anvikar AR. Prevalence of Pfhrp2/3 gene deletions among false negative rapid antigen test results in central India. J Vector Borne Dis 2021;58:273-80

How to cite this URL:
Kumari S, Ahmed MZ, Sharma S, Pande V, Anvikar AR. Prevalence of Pfhrp2/3 gene deletions among false negative rapid antigen test results in central India. J Vector Borne Dis [serial online] 2021 [cited 2023 Mar 29];58:273-80. Available from: http://www.jvbd.org//text.asp?2021/58/3/273/328815

  Introduction Top

Malaria is a life threating disease worldwide and is widely distributed across the India. The number of malaria cases in India were reduced in 2019; a 22% decrease compared to 2018 burden[1],[2]. In India, 90% of the estimated malaria cases were accorded only for 7 out of 36 states of the country with Chhattisgarh state being among them[1]. Chhattisgarh is a heavily forested state in the east central India accounting for nearly18% of the total malaria burden of India. This state alone is accountable for more than 31% of total P. falciparum malaria burden and 34% attributed death percentage in the country in 2019[2]. P. falciparum is the predominant (81%) parasite species, followed by P. vivax and is a major health problem of the state because of its large tribal population[3]. The diagnosis of malaria in India is primarily done by microscopy. However, malaria rapid diagnostic tests (RDTs) have become an important tool for diagnosis in remote areas where microscopy results would not be directly available or its use may not be feasible[4].

Histidine-rich protein 2 (PfHRP2) is produced during all blood stages (asexual phase) of the P. falciparum life cycle and hence serves as a good candidate for the RDTs. HRP2-based RDTs performance depends on various factors such as antigenic variability of the target protein, persistence of antigen in the bloodstream following elimination of parasites, quality of manufacture, storage conditions, parasite density below the RDT threshold of detection[5]. The sensitivity of HRP2-based RDTs is also affected by deletion of genes targeted for antigens or due to change in protein expression or mutations like frame shift mutation. PfHRP2 based RDTs which recognizes the specific parasitic diagnostic antigen, may also produce false negative result due to variability of this antigen and gene deletion phenomenon. There are reports of in vitro adapted parasitic lines lacking either or both Pfhrp2 or Pfhrp3 genes and in the progeny of a genetic cross[6].

Previous study of P. falciparum infected samples from Peru, Mali, Brazil, Colombia, Honduras, Guyana, Bangladesh, China Myanmar border, Ghana, Congo, French Guiana, Eritrea, Kenya, Mozambique and India have reports of lacking one or both of Pfhrp2/Pfhrp- 3genes which were positive by microscopy but negative with PfHRP2-detecting RDTs[4],[7],[8],[9],[10],[11],[12],[13]. False negative results produced by malaria RDTs may lead to misdiagnosis and failure of proper treatment of patients.

Low level prevalence of Pfhrp2 and Pfhrp3 deleted P. falciparum parasites were reported from most of the endemic areas of India, and these are recommended for intermittent surveillance for consistent use of PfHRP2 based RDTs. However, Odisha state reported a high incidence of Pfhrp2 (9.89%) and Pfhrp3 (6.25%) gene deletions[14]. Based on the above reports, the present study was aimed to check the Pf-HRP2 RDT false negative scenario in the context of parasite density and Pfhrp2/ Pfhrp3 gene deletion.

  Material & Methods Top

Study site and sample collection

Suspected malaria cases blood samples were collected from districts Dhamtari (20°7015’ N latitude and 81°5542’ E longitude), Kanker (20°2675’ N latitude and 81°4927’ E longitude), and Raipur (21°2514› N latitude and 81°6296› E longitude) of Chhattisgarh state during September 2016 to October 2017. Two hundred and three samples were screened for malaria. Blood samples were drawn by the finger prick method and used for slide preparation, RDT performance and DBS (Dried Blood Spots) preparation. All patients were treated with antimalarial drugs as per the National Drug Policy.

Microscopy and RDT

Thick and thin blood film was prepared, air dried and stained by Giemsa after methanol fixation. The slides parasite density was calculated as number of parasites/μL= total number of asexual parasites/200 WBC × 8000. Two different PfHRP2 antigen based RDT kits, i.e. Falcivax (Zephyr Biomedicals, India) and SD Malaria AgPf/Pv (Alere Medical Pvt. Ltd, India) performance were tested for microscopy confirmed P. falciparum malaria samples. In case of disparity among suspected cases, RDT tests were repeated twice.

Genomic DNA isolation

Three punches each of 1mm diameter size of DBS filter paper spots were taken for the genomic DNA isolation using QIAmp DNA blood mini kit (QIAGEN, Valencia, CA) in accordance with the manufacturer’s protocol. The extracted DNA was quantified using NanoDrop spectro-photometer (Thermo Scientific, USA) and stored at -20°C till use.

Diagnostic PCR

Using 18sRNA gene PCR amplification, P. falciparum infections were confirmed. Before processing for molecular analysis for the presence of Pfhrp2 /Pfhrp3 genes and its flanking regions we first checked the intactness of DNA by PCR amplification for the three single copy genes Pfmsp1, Pfmsp2and Pfglurp using methods described elsewhere[15]. The samples qualified with successful amplification for these three genes were further tested for Pfhrp2/Pfhrp2 gene deletions.

Pfhrp2 and Pfhrp3 genes detection by PCR

PCR was performed for the confirmation of Pfhrp2 and Pfhrp3 gene deletion by amplifying regions of exon2 and across exon1-2. Pfhrp2 exon2 and Pfhrp3 exon2 were amplified using primers and cyclic conditions described in publication[16] while Pfhrp2 across exon1-2 and Pfhrp3 across exon 1-2 amplification were performed in accordance to the method described earlier with some modifications in cyclic conditions[5],[17].

Pfhrp2 and Pfhrp3 neighboring genes detection by PCR

Pfhrp2 upstream gene (PF3D7_0831900): 5.535 kb upstream, Pfhrp2 downstream gene (PF3D7_0831700): 6.49 kb downstream, Pfhrp3 upstream (PF3D7_1372100): 4.404 kb upstream and Pfhrp3 downstream(PF3D7_1372400): 1.68kb downstream genes were amplified using standard primers with minor modifications in cyclic conditions [Table S1][5].PCR reactions were performed for 25μl per reaction volume. Amplified products were resolved on 2% agarose gel and images were capturedunder UVITEC gel documentation system in UV light and specific band length wasidentified using 100bp molecular ladder.

Validation of gene deletion and estimation of parasitaemia by qPCR

Real-time PCR (Light Cycler 480 system, Roche) was performed for the validation of gene deletion and parasitaemia estimation in 20μl per reaction volume using 96- well plate (Bio-Rad, USA). Reaction mixture contained 2X SYBR green PCR master mixes (KAPA Biosystems, USA) and 0.25μM each of forward and reverse primers as per method described elsewhere[17]. DNase, RNase and Protease free molecular grade water (CELL Clone, Genet-ix, India) was used for volume adjustment and in making dilutions. P. falciparum 3D7 laboratory strain was used to derive the standard curve for estimation of parasitaemia of the respective samples and also used as positive control while P. falciparum Dd2 laboratory strain was used as negative control wherever applicable. All qPCR experiments were performed in replicates. Primers and PCR conditions are mentioned in [Table S1] with slightly modified protocol.

Ethical statement

The study was approved by the Institutional Ethics Committee of the ICMR-National Institute of Malaria Research, New Delhi (Vide Letter No. ECR/NIMR/ EC/2012/38). The study was also approved by the Institutional Publication committee of ICMR-National Institute of Malaria Research, New Delhi (PSC Approval No. 62/2019).

  Results Top

Out of total 203 samples screened, 85 cases were found P. falciparum malaria positive by microscopy and PCR. 81 samples were found PfHRP2-RDT positive and four samples (CKAG45, CKAG152, CKAG332 and CKAG209) were observed PfHRP2-RDT negative. Three samples were found P. vivax malaria positive and two samples were positive for both P. falciparum and P. vivax species among 203 samples. For all P. falciparum malaria patients, median (Interquartile ranges) and frequency (percentage) calculated for age was 24 (9-34) among which 43% were female and parasitaemia calculated was 14515 parasite/μl (1678.75–26212.25). Those four PfHRP2-RDT negative samples were further analyzed for Pfhrp2and Pfhrp3 gene deletions. The selection strategy for concluding the deletion of Pfhrp2 and Pfhrp3 genes are mentioned in [Figure 1].
Figure 1: Flow diagram presentation of sample selection strategy for Pfhrp2 and Pfhrp3 gene.

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Specificity and sensitivity of RDTs, microscopy and PCR

Among all the malaria cases which were confirmed for P. falciparum mono-infections, 4 samples were observed RDT negative but PCR and microscopically positive for P. falciparum infection. Of note, three different detection methods were used which presented a comprehensive outlook for all the samples. Among the three-detection method employed, sensitivity and specificity of PfHRP2-RDT observed were 95% while for PCR it was found 100% with respect to gold standard microscopy in P. falciparum positive samples. To exclude any possibility of misdiagnosis, those four PfHRP2-RDT negative and microscopy positive samples were diagnosed twice with two different RDT kits and every time they produced negative result for PfHRP2 antigen.

Species identification and confirmation of intact DNA quality

The DNA samples were PCR amplified using primers specific for 18srRNA gene to confirm P. falciparum specific malaria. All 85 samples which were RDT tested or microscopically confirmed for P. falciparum malaria were further verified by Nested PCR for P. falciparum mono infection. Co-infection cases were excluded from the study. Following species confirmation, quality and intactness of all the samples were checked for three specific genes i.e., Pfmsp1, Pfmsp2 and Pfglurp [Figure 2]A,[Figure 2]B,[Figure 2]C. All confirmed P. falciparum positive samples were found intact.
Figure 2: Amplifications of Plasmodium falciparum genes (A) Pfglurp, (B) Pfmsp2 and (C) Pfmsp1 showing DNA intactness resolved on 2% agarose gel. (D-G) Pfhrp2 and neighbouring genes. (H-K) Pfhrp3 and neighbouring genes. Lane1=100bp molecular marker, Lane 2 and 3=3D7 & Dd2 strain. Lane 4,5,6&7=field samples (CKAG45, CKAG152, CKAG332 and CKAG209) of gene deletion. Lane8=negative control.

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Prevalence of Pfhrp2/Pfhrp3 genes and its neighboring gene deletion

The four samples of Kanker district (CKAG45, CKAG152, CKAG332 and CKAG209) were subjected to Pfhrp2exon 2 specific PCR amplification and analysis. It was observed that except for the sample CKAG209, the other samples (CKAG45, CKAG152, CKAG332) did not yield any product and were diagnosed as lack of Pfhrp2exon2 [Figure 2]D compared to 3D7 positive control and similar to Dd2 negative control, a Pfhrp2 null strain. The positivity of Pfhrp2exon2 for sample CKAG209 while been negative in RDT spotted a ground to check the presence/absence of Pfhrp3 exon2 as well for all the samples. Similarly, it was observed that the respective samples (CKAG45, CKAG152, CKAG332) did not produce any desired PCR amplified product for Pfhrp3 exon2 and were diagnosed as Pfhrp3 exon2 deleted except for the sample CKAG209 [Figure 2]H. Pfhrp2 and Pfhrp3 neighboring genes PCR amplification were further performed to check their possible association with the RDT target region (Pfhrp2exon2) in the samples CKAG45, CKAG152, CKAG332 and CKAG209. Samples CKAG45, CKAG152 and CKAG332 failed to produce any desired PCR amplified product for Pfhrp2 upstream [Figure 2]E, Pfhrp2 downstream [Figure 2]F and Pfhrp2 exon 1-2 [Figure 2]G and were concluded deleted from the respective samples. Further, Pfhrp3upstream [Figure 2]I, Pfhrp3 downstream [Figure 2]J and Pfhrp3 exon 1-2 [Figure 2]K did not yield any PCR amplification as well and were diagnosed as deleted from the CKAG45, CKAG152 and CKAG332 samples. In either case, sample CKAG209 showed amplification for Pfhrp2/3 exon2, Pfhrp2/3 upstream and Pfhrp2/3 downstream genes. Conclusively, out of four, three samples were diagnosed as complete deletion for the Pfhrp2 and Pfhrp3 and its neighboring genes while one was diagnosed positive for all respective genes.

Validation of complete deletion by qPCR and estimation of P. falciparum parasitemia

For further validation and Pfhrp2 and Pfhrp3 genes complete deletion confirmation, Pfhrp gene specific qPCR analysis were performed using 3D7 laboratory strain as positive control while a laboratory confirmed mono-infected P.vivax DNA was used as negative control. The purpose of taking P. vivax as a negative control was that it completely lacks the Histidine Rich Protein (HRP protein) due to the lack of hrp gene sequences. It was observed that sample CKAG209 though displayed a higher peak than 3D7 laboratory strain, revealed a similar Tm value range of 76°C to 82.5°C, therefore confirming the presence of Pfhrp gene [Figure 3]A & [Figure 3]B whereas samples CKAG45, CKAG152 and CKAG332 exhibited a Tm value range between 86°C to 90°C similar to displayed by P.vivax negative control, authenticating the absence of Pfhrp gene. Thus, combining all the results obtained [Table 1]; samples CKAG45, CKAG152 and CKAG332 showed a complete deletion state validated by lack of Pfhrp2 and Pfhrp3 exon2 genes whereas sample CKAG209 displayed presence of Pfhrp2 and Pfhrp3 exon2 genes and substantiated that it is not a Pfhrp2 and Pfhrp3 deletion case though negative for PfHRP2-RDT. Furthermore, to conclude whether performance of RDT was affected by the parasitaemia factor, parasite quantification was performed for the samples CKAG45, CKAG152, CKAG332 and CKAG209 by qPCR using Pfcytb gene copy number estimation method[17]. Using published genome size for P. falciparum (22.8Mbp), it was calculated that 1ng of P. falciparum DNA is equal to 40.6× 103genome copies[18]. In the present study, 7ng of P. falciparum 3D7 strain was used to derive the standard curve for parasitaemia estimation [Figure 4]. Using parasitaemia standard curve and Ct value obtained for the respective samples, it was observed that the evaluated copy number obtained for the sample CKAG209 was greater than 2.8×105parasite/μl, whereas for Pfhrp2 and Pfhrp3 gene deleted samples CKAG45, CKAG152 and CKAG332, the estimated copy number observed was lesser than the value 277 parasite/ μL [Table 2]. Thus, evidently sample CKAG209 exhibited a PfHRP2-RDT false negative status as a result of higher parasite density whilst three samples CKAG45, CKAG152 and CKAG332 revealed a Pfhrp2 and Pfhrp3 complete deletion and low parasitaemia state.
Figure 3: qPCR showing amplified melting peaks (A) and melting curves (B) of Pfhrp gene in respected samples. (a) & (b) Field isolate sample CKAG209 showed a high peak melting curve (76°C to 82.5°C) similar to 3D7 which was used as positive control. (c), (d) & (f) Field isolate samples CKAG152, CKAG45 and CKAG332 showed smaller peak of value 86°C to 90°C and similar to P. vivax negative control; confirms absence of Pfhrp specific melting peaks in a Pfhrp specific primers-based qPCR experiment. (e) P. vivax as negative control. (g) NTC; molecular grade water.

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Figure 4: Standard curve generated for the quantification of P. falciparum parasitaemia in respective samples. 3D7 P. falciparum laboratory strain dilutions (1:1 ratio) were used to derive the equation.

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Table 1: RDTs and Gene amplification results by PCR and Real-time PCR of Pfhrp2 and Pfhrp3 and its neighboring genes.

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  Discussion Top

The explanation for PfHRP2-RDT false negative consequence in this study was attributed primarily due to Pfhrp2/3 gene deletions, however, the other potential causes i.e. Pfhrp3 antigens cross reactivity, low expression of the HRP2 antigen, target epitope amino acid sequence variations, very low or high parasite density or poor RDT quality and sensitivity may also contribute in the RDT false negative outcome[19]. For higher sensitivity and specificity of the HRP2-RDT, lower parasite density recommended is 200 parasite/μl; though, reasonable HRP2-RDT performance could also be obtained with confidence for above 100 parasite/μl[20]. On the other side, higher parasitic load affect the performance of HRP2-RDT and could result in false negative situation or prozone-effect like phenomenon which occurs when high concentrations of antibody is been saturated by antigen and resulted in prevention of lattice formation, hence loss in antigen-antibody precipitation[19],[21]. Moreover, in this report, four samples were HRP2-RDT false negative and among them three samples were diagnosed Pfhrp2and Pfhrp3 (exon2 and across exon 1-2 regions) deleted besides lacking its immediate flanking regions which is liable for 3.5% overall deletion frequency of the state. Lower parasitaemia association was observed with the complete deletions of Pfhrp2 and Pfhrp3 genes displayed by the respective samples (CKAG45, CKAG152 and CKAG332). Moreover, sample CKAG209, displayed HRP2-RDT false negative due to higher parasite density (prozone effect) considered as an important factor affecting RDT performance. Escaping RDT diagnosis through Pfhrp2and Pfhrp3 complete gene deletion and keeping lower parasite density in host could be described as remarkable survival strategy of the parasite for an efficient transmission. RDT false negative circumstances either due to Pfhrp2/3 gene deletion or prozone effect could result in underprivileged malaria diagnosis and hence may aid in escaping of parasite from anti-malarial treatments and inefficient malaria elimination efforts in developing and high-risk countries. Although the first Pfhrp2/3 deletion report from India revealed a moderate to high parasitaemia range of approx. 6952 parasite/μl and 47,136 parasite/μl respectively[12], lower parasitaemia incidence were also been reported where 52% Pfhrp2/3 deletion cases had <500 parasite/μl (24 out of 46) and amongst that 41.6% reports had <200 parasite/μl (10 out of 24)[8]. The worldwide surveillance of Pfhrp2/Pfhrp3 deletion is summarized in [Table S2] which reflects the global spread situation of Pfhrp2/Pfhrp3gene deletions. Though none of the reports elucidate any direct association of Pfhrp2/Pfhrp3 deletion with that of the parasite density, several Pfhrp2/Pfhrp3 deletion cases revealed a lower parasite level[10],[22],[23],[24] similar to the present study.

Low parasitic load association with the Pfhrp2/Pfhrp3 complete gene deletion situation though not always factual, its association cannot be ruled out as it may be helpful in its efficient transmission. However, it is evident from previous studies that random deletion of Pfhrp2 and Pfhrp3 may be a natural phenomenon i.e. random presence of deleted parasites in the populations[5],[7]. Concerning the high deletion frequencies in some diverse areas like in Peru[5], Colombia[24], Honduras (Pfhrp3)[4] and Eritrea[9], the spread of Pfhrp2/Pfhrp3 deletions in the present study, may highlight the problem of increasing gene deletion cases of the parasite as reported in Korea district, Chhattisgarh (deletion frequency as high as 25%) suggests continuous monitoring of Pfhrp2 deletion[8].

The present study was a pilot study with small sample size focused on to tracking the problem of misdiagnosis due to RDT false negative performance in endemic rural areas. The RDT failure in P. falciparum diagnosis in this study was due to untraceable HRP2 antigen. The loss of RDT functionality was due to the absence of Pfhrp2 and Pfhrp3 antigens which was further identified as complete deletion due to the lack of their neighboring genes. Higher parasite level was also found affecting the RDT performance by producing prozone effect while complete Pfhrp2/Pfhrp3 deletion samples had lower parasitaemia. Moreover, PfHRP2-RDT performance was observed to be compromised with the Pfhrp2/3 gene deletions and are associated with false negative results, hence, improper malaria misdiagnosis and failed treatment. Parasite persistent in host either with low or high concentrations may cause serious public health concern or pathological conditions due to alterations in immune factors[25]. Further studies on misdiagnosis due RDT false negative in context of Pfhrp2/3 gene deletion and parasitaemia in large sample size will be more beneficial to the malaria case control program and elimination.

  Conclusion Top

This study revealed a pervasiveness of PfHRP2-RDT false negative cases in the rural areas of Chhattisgarh, India, due to Pfhrp2/Pfhrp3 gene deletions. Pfhrp2 and Pfhrp3 deletions that result in false negative RDTs were uncommon in our setting. The continued monitoring of RDTS which results in false negative tests due Pfhrp2/3 gene deletion is the need of the hour for an effective malaria elimination strategy.

Conflict of interest: None

Supplementary Table 1

[Additional file 1]

Supplementary Table 2

[Additional file 2]

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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