Modelling the dynamics of Plasmodium falciparum histidine-rich protein 2 in human malaria to better understand malaria rapid diagnostic test performance

BACKGROUND: Effective diagnosis of malaria is a major component of case management. Rapid diagnostic tests (RDTs) based on Plasmodium falciparumhistidine-rich protein 2 (PfHRP2) are popular for diagnosis of this most virulent malaria infection. However, concerns have been raised about the longevity of the PfHRP2 antigenaemia following curative treatment in endemic regions. METHODS: A model of PfHRP2 production and decay was developed to mimic the kinetics of PfHRP2 antigenaemia during infections. Data from two human infection studies was used to fit the model, and to investigate PfHRP2 kinetics. Four malaria RDTs were assessed in the laboratory to determine the minimum detectable concentration of PfHRP2. RESULTS: Fitting of the PfHRP2 dynamics model indicated that in malaria naive hosts, P. falciparum parasites of the 3D7 strain produce 1.4 x 10(-)(1)(3) g of PfHRP2 per parasite per replication cycle. The four RDTs had minimum detection thresholds between 6.9 and 27.8 ng/mL. Combining these detection thresholds with the kinetics of PfHRP2, it is predicted that as few as 8 parasites/muL may be required to maintain a positive RDT in a chronic infection. CONCLUSIONS: The results of the model indicate that good quality PfHRP2-based RDTs should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days after treatment. The duration of a positive test result following curative treatment is dependent on the duration and density of parasitaemia prior to treatment and the presence and affinity of anti-PfHRP2 antibodies.

Malaria rapid diagnostic test performance : results of WHO product testing of malaria RDTs : round 5 (2013)

WHO estimates that half the world’s population is at risk of malaria. In 2012, there were an estimated 207 million cases (with an uncertainty range of 135 million to 287 million) and an estimated 627 000 deaths (with an uncertainty range of 473 000 to 789 000). Approximately 90% of all malaria deaths occur in sub-Saharan Africa, and 77% occur in children under 5 years. Malaria remains endemic in 104 countries, and, while parasite-based diagnosis is increasing, most suspected cases of malaria are still not properly confirmed, resulting in over-use of antimalarial drugs and poor disease monitoring (1)...

Malaria rapid diagnostic test performance: Results of WHO product testing of malaria RDTs: Round 6 (2014-2015)

Overview This report, published in conjunction with a summary overview of results of rounds 1–6, is the sixth in a series of laboratory-based evaluations of rapid diagnostic tests (RDTs) for malaria. It provides a comparative measure of their performance in a standardized way to distinguish between well and poorly performing tests. It can be used by malaria control programmes and guide WHO procurement recommendations for these diagnostic tools. The evaluation reported here was a joint project of the WHO Global Malaria Programme, the Foundation for Innovative New Diagnostics (FIND) and the United States Centers for Disease Control and Prevention (CDC) within the WHO-FIND Malaria RDT Evaluation Programme. The project was financed by FIND through a grant from UNITAID.

Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains : implications for rapid diagnostic tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

Blood transfer devices for malaria rapid diagnostic tests : evaluation of accuracy, safety and ease of use

Background: Malaria rapid diagnostic tests (RDTs) are increasingly used by remote health personnel with minimal training in laboratory techniques. RDTs must, therefore, be as simple, safe and reliable as possible. Transfer of blood from the patient to the RDT is critical to safety and accuracy, and poses a significant challenge to many users. Blood transfer devices were evaluated for accuracy and precision of volume transferred, safety and ease of use, to identify the most appropriate devices for use with RDTs in routine clinical care. Methods: Five devices, a loop, straw-pipette, calibrated pipette, glass capillary tube, and a new inverted cup device, were evaluated in Nigeria, the Philippines and Uganda. The 227 participating health workers used each device to transfer blood from a simulated finger-prick site to filter paper. For each transfer, the number of attempts required to collect and deposit blood and any spilling of blood during transfer were recorded. Perceptions of ease of use and safety of each device were recorded for each participant. Blood volume transferred was calculated from the area of blood spots deposited on filter paper. Results: The overall mean volumes transferred by devices differed significantly from the target volume of 5 microliters (p < 0.001). The inverted cup (4.6 microliters) most closely approximated the target volume. The glass capillary was excluded from volume analysis as the estimation method used is not compatible with this device. The calibrated pipette accounted for the largest proportion of blood exposures (23/225, 10%); exposures ranged from 2% to 6% for the other four devices. The inverted cup was considered easiest to use in blood collection (206/ 226, 91%); the straw-pipette and calibrated pipette were rated lowest (143/225 [64%] and 135/225 [60%] respectively). Overall, the inverted cup was the most preferred device (72%, 163/227), followed by the loop (61%, 138/227). Conclusions: The performance of blood transfer devices varied in this evaluation of accuracy, blood safety, ease of use, and user preference. The inverted cup design achieved the highest overall performance, while the loop also performed well. These findings have relevance for any point-of-care diagnostics that require blood sampling.

Circulating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests

Background Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. Methods The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. Results Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. Conclusions These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.

Pan-Plasmodium band sensitivity for Plasmodium falciparum detection in combination malaria rapid diagnostic tests and implications for clinical management

Background: Malaria rapid diagnostic tests (RDTs) are appropriate for case management, but persistent antigenaemia is a concern for HRP2-detecting RDTs in endemic areas. It has been suggested that pan-pLDH test bands on combination RDTs could be used to distinguish persistent antigenaemia from active Plasmodium falciparum infection, however this assumes all active infections produce positive results on both bands of RDTs, an assertion that has not been demonstrated. Methods: In this study, data generated during the WHO-FIND product testing programme for malaria RDTs was reviewed to investigate the reactivity of individual test bands against P. falciparum in 18 combination RDTs. Each product was tested against multiple wild-type P. falciparum only samples. Antigen levels were measured by quantitative ELISA for HRP2, pLDH and aldolase. Results: When tested against P. falciparum samples at 200 parasites/μL, 92% of RDTs were positive; 57% of these on both the P. falciparum and pan bands, while 43% were positive on the P. falciparum band only. There was a relationship between antigen concentration and band positivity; ≥4 ng/mL of HRP2 produced positive results in more than 95% of P. falciparum bands, while ≥45 ng/mL of pLDH was required for at least 90% of pan bands to be positive. Conclusions: In active P. falciparum infections it is common for combination RDTs to return a positive HRP2 band combined with a negative pan-pLDH band, and when both bands are positive, often the pan band is faint. Thus active infections could be missed if the presence of a HRP2 band in the absence of a pan band is interpreted as being caused solely by persistent antigenaemia.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

The development of a serological-based diagnostic test for Dasheen mosaic potyvirus (DsMV)

Dasheen mosaic potyvirus (DsMV) is an important virus affecting taro. The virus has been found wherever taro is grown and infects both the edible and ornamental aroids, causing yield losses of up to 60%. The presence of DsMV, and other viruses,prevents the international movement of taro germplasm between countries. This has a significant negative impact on taro production in many countries due to the inability to access improved taro lines produced in breeding programs. To overcome this problem, sensitive and reliable virus diagnostic tests need to be developed to enable the indexing of taro germplasm. The aim of this study was to generate an antiserum against a recombinant DsMV coat protein (CP) and to develop a serological-based diagnostic test that would detect Pacific Island isolates of the virus. The CP-coding region of 16 DsMV isolates from Papua New Guinea, Samoa, Solomon Islands, French Polynesia, New Caledonia and Vietnam were amplified,cloned and sequenced. The size of the CP-coding region ranged from 939 to 1038 nucleotides and encoded putative proteins ranged from 313 to 346 amino acids, with the molecular mass ranging from 34 to 38 kDa. Analysis ofthe amino acid sequences revealed the presence of several amino acid motifs typically found in potyviruses,including DAG, WCIE/DN, RQ and AFDF. When the amino acid sequences were compared with each other and the DsMV sequences on the database, the maximum variability was21.9%. When the core region ofthe CP was analysed, the maximum variability dropped to 6% indicating most variability was present in the N terminus. Within seven PNG isolates ofDsMV, the maximum variability was 16.9% and 3.9% over the entire CP-coding region and core region, respectively. The sequence ofPNG isolate P1 was most similar to all other sequences. Phylogenetic analysis indicated that almost all isolates grouped according to their provenance. Further, the seven PNG isolates were grouped according to the region within PNG from which they were obtained. Due to the extensive variability over the entire CP-coding region, the core region ofthe CP ofPNG isolate Pl was cloned into a protein expression vector and expressed as a recombinant protein. The protein was purified by chromatography and SDS-PAGE and used as an antigen to generate antiserum in a rabbit. In western blots, the antiserum reacted with bands of approximately 45-47 kDa in extracts from purified DsMV and from known DsMV -infected plants from PNG; no bands were observed using healthy plant extracts. The antiserum was subsequently incorporated into an indirect ELISA. This procedure was found to be very sensitive and detected DsMV in sap diluted at least 1:1,000. Using both western blot and ELISA formats,the antiserum was able to detect a wide range ofDsMV isolates including those from Australia, New Zealand, Fiji, French Polynesia, New Caledonia, Papua New Guinea, Samoa, Solomon Islands and Vanuatu. These plants were verified to be infected with DsMV by RT-PCR. In specificity tests, the antiserum was also found to react with sap from plants infected with SCMV, PRSV-P, PRSV-W, but not with PVY or CMV -infected plants.

Identification of optimal epitopes for Plasmodium falciparum rapid diagnostic tests that target histidine-rich proteins 2 and 3

Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs.

Ongoing outbreak of dengue serotype 3 in Solomon Islands, January to May 2013

Introduction In January 2013, clinicians in Honiara, Solomon Islands noted several patients presenting with dengue-like illness. Serum from three cases tested positive for dengue by rapid diagnostic test. Subsequent increases in cases were reported, and the outbreak was confirmed as being dengue serotype-3 by further laboratory tests. This report describes the ongoing outbreak investigation, findings and response. Methods Enhanced dengue surveillance was implemented in the capital, Honiara, and in the provinces. This included training health staff on dengue case definitions, data collection and reporting. Vector surveillance was also conducted. Results From 3 January to 15 May 2013, 5254 cases of suspected dengue were reported (101.8 per 10 000 population), including 401 hospitalizations and six deaths. The median age of cases was 20 years (range zero to 90), and 86% were reported from Honiara. Both Aedes aegyti and Aedes albopictus were identified in Honiara. Outbreak response measures included clinical training seminars, vector control activities, implementation of diagnostic and case management protocols and a public communication campaign. Discussion This was the first large dengue outbreak documented in Solomon Islands. Factors that may have contributed to this outbreak include a largely susceptible population, the presence of a highly efficient dengue vector in Honiara, a high-density human population with numerous breeding sites and favourable weather conditions for mosquito proliferation. Although the number of cases has plateaued since 1 April, continued enhanced nationwide surveillance and response activities are necessary.

A 2-hour diagnostic protocol for possible cardiac chest pain in the emergency department: A randomized clinical trial

IMPORTANCE Patients with chest pain represent a high health care burden, but it may be possible to identify a patient group with a low short-term risk of adverse cardiac events who are suitable for early discharge. OBJECTIVE To compare the effectiveness of a rapid diagnostic pathway with a standard-care diagnostic pathway for the assessment of patients with possible cardiac chest pain in a usual clinical practice setting. DESIGN, SETTING, AND PARTICIPANTS A single-center, randomized parallel-group trial with blinded outcome assessments was conducted in an academic general and tertiary hospital. Participants included adults with acute chest pain consistent with acute coronary syndrome for whom the attending physician planned further observation and troponin testing. Patient recruitment occurred from October 11, 2010, to July 4, 2012, with a 30-day follow-up. INTERVENTIONS An experimental pathway using an accelerated diagnostic protocol (Thrombolysis in Myocardial Infarction score, 0; electrocardiography; and 0- and 2-hour troponin tests) or a standard-care pathway (troponin test on arrival at hospital, prolonged observation, and a second troponin test 6-12 hours after onset of pain) serving as the control. MAIN OUTCOMES AND MEASURES Discharge from the hospital within 6 hours without a major adverse cardiac event occurring within 30 days. RESULTS Fifty-two of 270 patients in the experimental group were successfully discharged within 6 hours compared with 30 of 272 patients in the control group (19.3% vs 11.0%; odds ratio, 1.92; 95% CI, 1.18-3.13; P = .008). It required 20 hours to discharge the same proportion of patients from the control group as achieved in the experimental group within 6 hours. In the experimental group, 35 additional patients (12.9%) were classified as low risk but admitted to an inpatient ward for cardiac investigation. None of the 35 patients received a diagnosis of acute coronary syndrome after inpatient evaluation. CONCLUSIONS AND RELEVANCE Using the accelerated diagnostic protocol in the experimental pathway almost doubled the proportion of patients with chest pain discharged early. Clinicians could discharge approximately 1 of 5 patients with chest pain to outpatient follow-up monitoring in less than 6 hours. This diagnostic strategy could be easily replicated in other centers because no extra resources are required.

Optimisation of Banana streak virus (BSV) diagnostic assays

Bananas are one of the world�fs most important crops, serving as a staple food and an important source of income for millions of people in the subtropics. Pests and diseases are a major constraint to banana production. To prevent the spread of pests and disease, farmers are encouraged to use disease�] and insect�]free planting material obtained by micropropagation. This option, however, does not always exclude viruses and concern remains on the quality of planting material. Therefore, there is a demand for effective and reliable virus indexing procedures for tissue culture (TC) material. Reliable diagnostic tests are currently available for all of the economically important viruses of bananas with the exception of Banana streak viruses (BSV, Caulimoviridae, Badnavirus). Development of a reliable diagnostic test for BSV is complicated by the significant serological and genetic variation reported for BSV isolates, and the presence of endogenous BSV (eBSV). Current PCR�] and serological�]based diagnostic methods for BSV may not detect all species of BSV, and PCR�]based methods may give false positives because of the presence of eBSV. Rolling circle amplification (RCA) has been reported as a technique to detect BSV which can also discriminate between episomal and endogenous BSV sequences. However, the method is too expensive for large scale screening of samples in developing countries, and little information is available regarding its sensitivity. Therefore the development of reliable PCR�]based assays is still considered the most appropriate option for large scale screening of banana plants for BSV. This MSc project aimed to refine and optimise the protocols for BSV detection, with a particular focus on developing reliable PCR�]based diagnostics Initially, the appropriateness and reliability of PCR and RCA as diagnostic tests for BSV detection were assessed by testing 45 field samples of banana collected from nine districts in the Eastern region of Uganda in February 2010. This research was also aimed at investigating the diversity of BSV in eastern Uganda, identifying the BSV species present and characterising any new BSV species. Out of the 45 samples tested, 38 and 40 samples were considered positive by PCR and RCA, respectively. Six different species of BSV, namely Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV), Banana streak UA virus (BSUAV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), were detected by PCR and confirmed by RCA and sequencing. No new species were detected, but this was the first report of BSMYV in Uganda. Although RCA was demonstrated to be suitable for broad�]range detection of BSV, it proved time�]consuming and laborious for identification in field samples. Due to the disadvantages associated with RCA, attempts were made to develop a reliable PCR�]based assay for the specific detection of episomal BSOLV, Banana streak GF virus (BSGFV), BSMYV and BSIMV. For BSOLV and BSGFV, the integrated sequences exist in rearranged, repeated and partially inverted portions at their site of integration. Therefore, for these two viruses, primers sets were designed by mapping previously published sequences of their endogenous counterparts onto published sequences of the episomal genomes. For BSOLV, two primer sets were designed while, for BSGFV, a single primer set was designed. The episomalspecificity of these primer sets was assessed by testing 106 plant samples collected during surveys in Kenya and Uganda, and 33 leaf samples from a wide range of banana cultivars maintained in TC at the Maroochy Research Station of the Department of Employment, Economic Development and Innovation (DEEDI), Queensland. All of these samples had previously been tested for episomal BSV by RCA and for both BSOLV and BSGFV by PCR using published primer sets. The outcome from these analyses was that the newly designed primer sets for BSOLV and BSGFV were able to distinguish between episomal BSV and eBSV in most cultivars with some B�]genome component. In some samples, however, amplification was observed using the putative episomal�]specific primer sets where episomal BSV was not identified using RCA. This may reflect a difference in the sensitivity of PCR compared to RCA, or possibly the presence of an eBSV sequence of different conformation. Since the sequences of the respective eBSV for BSMYV and BSIMV in the M. balbisiana genome are not available, a series of random primer combinations were tested in an attempt to find potential episomal�]specific primer sets for BSMYV and BSIMV. Of an initial 20 primer combinations screened for BSMYV detection on a small number of control samples, 11 primers sets appeared to be episomal�]specific. However, subsequent testing of two of these primer combinations on a larger number of control samples resulted in some inconsistent results which will require further investigation. Testing of the 25 primer combinations for episomal�]specific detection of BSIMV on a number of control samples showed that none were able to discriminate between episomal and endogenous BSIMV. The final component of this research project was the development of an infectious clone of a BSV endemic in Australia, namely BSMYV. This was considered important to enable the generation of large amounts of diseased plant material needed for further research. A terminally redundant fragment (.1.3 �~ BSMYV genome) was cloned and transformed into Agrobacterium tumefaciens strain AGL1, and used to inoculate 12 healthy banana plants of the cultivars Cavendish (Williams) by three different methods. At 12 weeks post�]inoculation, (i) four of the five banana plants inoculated by corm injection showed characteristic BSV symptoms while the remaining plant was wilting/dying, (ii) three of the five banana plants inoculated by needle�]pricking of the stem showed BSV symptoms, one plant was symptomless while the remaining had died and (iii) both banana plants inoculated by leaf infiltration were symptomless. When banana leaf samples were tested for BSMYV by PCR and RCA, BSMYV was confirmed in all banana plants showing symptoms including those were wilting and/or dying. The results from this research have provided several avenues for further research. By completely sequencing all variants of eBSOLV and eBSGFV and fully sequencing the eBSIMV and eBSMYV regions, episomal BSV�]specific primer sets for all eBSVs could potentially be designed that could avoid all integrants of that particular BSV species. Furthermore, the development of an infectious BSV clone will enable large numbers of BSVinfected plants to be generated for the further testing of the sensitivity of RCA compared to other more established assays such as PCR. The development of infectious clones also opens the possibility for virus induced gene silencing studies in banana.

Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3 : a review and recommendations for accurate reporting

Malaria rapid diagnostic tests (RDTs) play a critical role in malaria case management, surveillance and case investigations. Test performance is largely determined by design and quality characteristics, such as detection sensitivity, specificity, and thermal stability. However, parasite characteristics such as variable or absent expression of antigens targeted by RDTs can also affect RDT performance. Plasmodium falciparum parasites lacking the PfHRP2 protein, the most common target antigen for detection of P. falciparum, have been reported in some regions. Therefore, accurately mapping the presence and prevalence of P. falciparum parasites lacking pfhrp2 would be an important step so that RDTs targeting alternative antigens, or microscopy, can be preferentially selected for use in such regions. Herein the available evidence and molecular basis for identifying malaria parasites lacking PfHRP2 is reviewed, and a set of recommended procedures to apply for future investigations for parasites lacking PfHRP2, is proposed.