Chapter 6 - Malaria diagnosis: Canadian recommendations for the prevention and treatment of malaria

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An Advisory Committee Statement (ACS) from the Committee to Advise on Tropical Medicine and Travel (CATMAT)

Updated 2023

Table of contents

• Preamble
• Summary
• Background
• Methods
• Microscopy
• Rapid diagnostic tests
• Nucleic acid amplification
• Table 6.1: Comparison of diagnostic tests for malaria
• Recommendations for malaria diagnostic testing
• Acknowledgements
• References

Preamble

The Committee to Advise on Tropical Medicine and Travel (CATMAT) provides the Public Health Agency of Canada (PHAC) with ongoing and timely medical, scientific, and public health advice relating to tropical infectious disease and health risks associated with international travel. PHAC acknowledges that the advice and recommendations set out in this statement are based upon the best current available scientific knowledge and medical practices, and is disseminating this document for information purposes to both travellers and the medical community caring for travellers.

Persons administering or using drugs, vaccines, or other products should also be aware of the contents of the product monograph(s) or other similarly approved standards or instructions for use. Recommendations for use and other information set out herein may differ from that set out in the product monograph(s) or other similarly approved standards or instructions for use by the licensed manufacturer(s). Manufacturers have sought approval and provided evidence as to the safety and efficacy of their products only when used in accordance with the product monographs or other similarly approved standards or instructions for use.

Summary

• The diagnosis of malaria is considered a medical emergency, and every clinical site where potential cases are assessed should have a system for ensuring rapid, reliable testing either on site or via rapid referral.
• Microscopy requires a high level of training and expertise, and remains the gold standard test.
• Rapid diagnostic tests, which are based on antigen detection, should always be promptly available. They have imperfect sensitivity, good specificity, but only moderately reliable speciation results.
• LAMP (a form of nucleic acid amplification test) is a rapid and simple test with high sensitivity and specificity; however it does not currently provide speciation. LAMP testing can be used to rule out malaria without further testing. The test is currently approved in Canada.
• Only microscopy can provide reliable speciation and parasitemia. Results are important for definitive management. All labs should endeavour to have a protocol for providing microscopy results within a few hours.
• PCR can provide very accurate speciation, especially for mixed infections, but is not commercialized and not easily or rapidly available for most centres in Canada.

Background

Plasmodium falciparum malaria can be rapidly fatal, particularly in a nonimmune host. It is the most urgent diagnosis to confirm or exclude in the febrile traveller who has been in a malaria-endemic zone. Signs and symptoms that may be present in people with malaria infection are neither sensitive nor specific. For example, fever is frequently not cyclic, and splenomegaly is rarely present early in the course of P. falciparum malaria ^{Footnote 1}. Hence, clinical assessment, even by experts, cannot reliably confirm or exclude a diagnosis of malaria ^{Footnote 2}.

The large majority of travel-related malaria cases diagnosed in nonendemic countries present within several months of return from an endemic area ^{Footnote 3}. However, some cases, including P. falciparum, will present after a longer period, that is, six months or more postexposure. For this reason, the CATMAT committee recommends that travellers who become ill with an unexplained fever within a year of returning home (regardless of whether malaria prophylaxis was prescribed or taken) should seek immediate medical attention and tell the physician their travel history. Particular attention should be paid to fevers that develop in the three months following travel, the period during which more than 90% of falciparum malaria manifests.

Health care providers should inform travellers of this advice as part of the pre-travel assessment, and physicians should include a travel history in the assessment of febrile patients.

Methods

This chapter was developed by a working group comprised of volunteers from the CATMAT committee. The working group, with support from the secretariat, was responsible for assessing available literature, synthesis and analysis of the evidence, drafting recommendations, and chapter writing. The final chapter was approved by the full CATMAT committee.

Recommendations were not developed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology. This decision, which was informed by guidance in the CATMAT statement on Evidence based process for developing travel and tropical medicine related guidelines and recommendations ^{Footnote 4}, was based on several considerations. Most recommendations in this chapter fall into the category of "good practice", in the sense that there is no reasonable alternative (e.g. testing for malaria should be done in a febrile traveller returning from an endemic area). In some cases, recommendations are made without a GRADE evaluation because there is significant benefit, with minimal potential harms (e.g. use of rapid diagnostic tests when malaria smears are not rapidly available). Finally, undertaking a GRADE-based approach to compare different diagnostic modalities was deemed as likely to require significant resources without materially affecting the nature of the recommendations. Consequently, recommendations in this chapter are based on a review and synthesis of the available evidence combined with expert opinion.

Microscopy

Confirmation or exclusion of a diagnosis of malaria depends upon laboratory examination of blood samples with parasitological tests. The standard test involves microscopic examination of thick and thin blood smears. Accurate examination of a blood smear requires considerable training and experience, particularly in the interpretation of the thick smear, parasite speciation and parasitemia quantification. Lack of experienced personnel may limit the accuracy of malaria diagnosis in laboratories in Canada ^{Footnote 5}, whereas diagnosis in low-income countries is further challenged by the quality of microscopy, and with supervision and quality control in the laboratory ^{Footnote 6}. For example, among US Peace Corps volunteers whose malaria was diagnosed by blood smear in local clinics in sub-Saharan Africa, the diagnosis could be confirmed in only 25% of cases ^{Footnote 7}.

When malaria is suspected, a Canadian laboratory should be able to confirm the presence of the parasite and, in most cases, identify the species within 2 hours of receiving a blood specimen ^{Footnote 5}. In very few cases, when the level of parasitemia is low, an initial smear and/or rapid test may be falsely negative. Thus, two additional smears should be examined at 6 to 12 hour intervals to confirm or exclude the diagnosis, or a rapid diagnostic test (RDT) should be performed (unless an alternate diagnosis can be firmly established). It is also important to obtain repeat smears at regular (usually at least daily) intervals to help evaluate disease evolution. There is no benefit in attempting to time blood sampling with the fever cycle, and this can potentially delay the diagnosis ^{Footnote 8}.

An essential element of interpreting malaria smears is the speciation of the parasite. Correct speciation may be critical to choosing the appropriate treatment and patient management. Quantitating parasitemia is also important for prognosticating, determining the need for parenteral treatment and the need for admission to an intensive care unit.

Rapid diagnostic tests

Rapid diagnostic tests (RDTs) do not require microscopy or specialized laboratory skills and can play a valuable adjunctive role in diagnosing malaria ^{Footnote 9}. A variety of RDTs are licensed by Health Canada for use in Canada and can be reviewed in the Medical Devices Active License Listing ^{Footnote 10}. RDTs are immunochromatographic assays that use monoclonal antibodies to capture malaria-specific antigens in a blood sample, producing a visible colour change. All tests include a positive control band that becomes visible as the sample migrates along the strip. The absence of a control band indicates an invalid test, but the presence of a visible control band does not assure reliability ^{Footnote 11}.

RDTs require small volumes of blood (2–50 μL) and can be done on fingerstick specimens or on anticoagulated blood or plasma. The current targets for RDTs are histidine-rich protein-2 of P. falciparum (PfHRP-2) or enzymes from the parasite glycolytic pathway, for example, species-specific lactate dehydrogenase (pLDH) or pan-species Plasmodium aldolase. Lactate dehydrogenase-based tests may detect all species of malaria (pan-specific) or may be species-specific for P. falciparum or P. vivax. Aldolase tests are pan-specific and cannot differentiate between any of the species of malaria.

The World Health Organization lists recommended RDTs that can be categorized into seven different types ^{Footnote 12}. Combinations of target antigens can be used to detect infection due to P. falciparum, P. vivax, mixed P. falciparum / P. vivax or mixed P. falciparum / non-falciparum species ^{Footnote 13}. To date, tests specific for P. malariae and P. ovale are not available ^{Footnote 14 Footnote 15}. P. knowlesi parasites cross-react with pLDH, and therefore tests may falsely report P. falciparum or P. vivax infection ^{Footnote 16 Footnote 17}. The sensitivity of most of these tests for P. falciparum is roughly comparable to microscopy, although they are not as sensitive as smears performed by expert microscopists. It is also important to understand that especially due to lack of sensitivity for all non-falciparum species, a negative RDT does not exclude malaria ^{Footnote 18}.

In Canada, RDTs are only approved for use in accredited laboratories. Although some RDTs were originally developed for use by travellers without access to effective malaria diagnosis, they have proved to be unreliable in this setting. Significant proportions of travellers are unable to complete the test procedure or interpret the results correctly ^{Footnote 19 Footnote 20} and the rates of false-negative results are unacceptable ^{Footnote 21}. In some cases, expatriates may be part of a reasonably stable community, which allows for the training of key members on the use of RDTs and the administration of appropriate self-treatment. Caution is warranted as there are few data from controlled studies on the use of RDTs in the long-term traveller or expatriate populations. An evaluation of a preventive malaria program for expatriates in malaria-endemic areas, which included RDTs and standby treatment, found that 15% of participants had difficulty performing the RDTs and 22% used standby treatment despite having a negative RDT ^{Footnote 22}. However, when used by trained laboratory staff, these tests can contribute to the rapid diagnosis of malaria pending confirmatory testing with microscopy and/or nucleic acid amplification tests ^{Footnote 9}.

In general, RDTs are best at detecting P. falciparum, with sensitivities between 88% and 100% and specificities between 92% and 96% ^{Footnote 23 Footnote 24}. A recent meta-analysis found that RDTs based on PfHRP-2 had a higher sensitivity but lower specificity in assessing for P. falciparum in high endemic regions compared with those based on pLDH (95.0% and 95.2% vs. 93.2% and 98.5%) ^{Footnote 15}. An earlier meta-analysis of RDTs in returned travellers found that tests based on PfHRP-2 were more accurate than those on pLDH ^{Footnote 9}. Sensitivity is decreased at parasite densities below 100/μL, with sensitivities of less than 70% at densities less than 50/μL ^{Footnote 25}. The sensitivity for the detection of P. vivax is inferior to that of P. falciparum ^{Footnote 19 Footnote 26}. For P. vivax, the data are limited, but the recommended threshold for satisfactory detection of parasitemia should be at least 200 parasites/μL ^{Footnote 27}.

Since the most common tests combine an aldolase- or pLDH band with an PfHRP-2 band a positive reaction may occur on both bands when only P. falciparum infection is present, making accurate diagnosis of mixed infections impossible without further testing ^{Footnote 23}. Geographic variation between P. falciparum strains could also affect test sensitivity ^{Footnote 28}.

RDTs are not recommended for assessing the response to antimalarial therapy. PfHRP-2 persists for prolonged periods after the asexual stage parasites (i.e., stages that are clinically relevant) have been cleared from blood by treatment, with 68% positivity at seven days and 27% positivity at 28 days after initiation of therapy ^{Footnote 25}. In addition to slow clearance of parasite antigen leading to false positive PfHRP-2 RDT, isolated sexual stage parasitemia (i.e., gametocytemia), can occur following successful treatment of malaria, and lead to a positive RDT result ^{Footnote 29}. Isolated gametocytemia following treatment of malaria does not warrant further treatment. The advantage of RDTs is that they are simple to use, provide rapid results, require no equipment and can be performed by laboratory staff who are untrained in malaria microscopy. However, results can be inaccurate if instructions are not followed carefully. Results must be read within the time frame specified by the manufacturer, as test lines may become positive after this time even in the absence of parasitemia. Heat and humidity can damage the tests, so test packages must be stored according to instructions and should only be opened immediately before use.

The presence of autoantibodies, such as rheumatoid factor, heterophile antibodies and anti-mouse antibodies, has been shown to give false-positive results in some test kits. The likelihood of a false-positive result in the presence of rheumatoid factor varies with the test antibody. Other infections that may cause fever in the returned traveller, such as dengue fever, African trypanosomiasis, toxoplasmosis, and tuberculosis, may lead to false positive RDTs ^{Footnote 18}. Occasional reports of negative RDTs in people with high levels of parasitemias are likely due to a "prozone" effect in which an excess of antigen masks the test antibody ^{Footnote 9 Footnote 19}.

There are increasing reports of false negative results for P. falciparum from multiple geographic regions due to strains genetically deficient in HRP-2 ^{Footnote 30 Footnote 31 Footnote 32 Footnote 33 Footnote 34}. The recent global emergence of Plasmodium falciparum strains with gene deletions or mutations of the histidine-rich protein 2 and 3 (HRP-2/3) genes has complicated interpretation of malaria antigen rapid diagnostic tests (RDTs) ^{Footnote 35}. The most common antigens for P. falciparum detection in RDTs are the HRP-2 protein and specific lactate dehydrogenase (Pf-pLDH) ^{Footnote 36}. HRP-3 shares significant homology with HRP-2, thus mutations in either gene may also affect sensitivity of RDTs utilizing HRP-2 as the target antigen ^{Footnote 37}. Because these mutations may result in reduced or no expression of HRP-2, HRP-3 or both, infections with these P. falciparum strains may demonstrate a negative or weak HRP-2 result on the PfHRP-2 RDTs assays. This may occur despite high parasitemia ^{Footnote 35}. At present, the pan-species antigens frequently used in combination with PfHRP-2 in these assays (pan-species proteins, such as lactate dehydrogenase (pan-pLDH) or aldolase) and the Plasmodium falciparum-specific LDH remains unaffected by these mutations. These mutations may have a significant impact on interpretation of RDT results. Assays that utilize only PfHPR-2 for the selective detection of P. falciparum may be falsely negative and those that also detect a pan-malarial antigen in combination with HRP-2/3 may result in false-identification of a non-falciparum infection.

HRP-2/3 deletion mutations have been reported out of South America (Peruvian Amazon Basin ^{Footnote 30}, Brazil ^{Footnote 38}), Africa (Senegal ^{Footnote 39}), Ethiopia ^{Footnote 37 Footnote 40}, Eritrea ^{Footnote 41}, Sudan ^{Footnote 42} and Kenya ^{Footnote 43} and India ^{Footnote 37}) and may represent a significant proportion of P. falciparum depending on the region ^{Footnote 44}.

Because of the potential risks associated with misdiagnosis of falciparum malaria CATMAT recommends that all patients suspected of having malaria who have a negative HRP2-based RDT test for Plasmodium falciparum have samples tested by an alternative method such as microscopy or nucleic acid amplification. Patients with a positive pan-malarial antigen and a negative HRP-2 test should have additional testing to definitively rule out P. falciparum malaria.

Nucleic acid amplification

Nucleic acid amplification (NAAT) assays for the detection of Plasmodium sp. directly from whole blood are highly sensitive (1 - 20 parasites/mL) ^{Footnote 45}, and where available with rapid turnaround time, may replace the need for multiple thick and thin smears for the diagnosis of malaria ^{Footnote 46 Footnote 47 Footnote 48 Footnote 49 Footnote 50}. Traditional PCR-based amplification methods are not commercialized in Canada, have limited availability, and are generally not available for rapid diagnosis, making them unsuitable for routine diagnosis. However, the regulatory approval of commercialized semi-automated rapid amplification assays for the detection of Plasmodium sp. DNA have resulted in more widespread availability in Canadian laboratories, including in rural areas traditionally lacking expertise in microscopy. Currently, the only licensed commercialized semi-automated rapid nucleic acid amplification assay in Canada is a loop-mediated isothermal amplification (LAMP) assay (Alethia Malaria Plus, Meridian Bioscience, formerly Illumigene Malaria). The equipment is simple to use and maintain, and requires minimal technical expertise. Studies have demonstrated that this assay is equivalent or superior in sensitivity to multiple thick smears and that a single assay can replace multiple smears as an initial diagnostic test, obviating the requirement for microscopic evaluation of multiple samples to rule out malaria infection in returning travellers. Reported sensitivity and specificity approach 100% in studies arbitrated by conventional PCR and microscopy ^{Footnote 46 Footnote 47 Footnote 48 Footnote 49 Footnote 50}. However, multiple limitations need to be considered when using these assays. This LAMP assay does not differentiate malaria species nor provide quantitation of malaria parasites, and does not distinguish residual nucleic acid versus intact parasites, i.e. may remain positive for up to three weeks after successful treatment. Thus, all positive specimens require follow-up thick and thin smears to provide speciation and quantitation for clinical management of patients ^{Footnote 47 Footnote 48}. Therefore, local expertise in microscopy or a robust and rapid referral system for speciation and quantitation must be maintained when utilizing commercial LAMP NAAT for the diagnosis of malaria. The persistence of Plasmodium DNA after treatment and isolated gametocytemia requires careful interpretation and correlation with microscopy and clinical presentation.

Table 6.1: Comparison of diagnostic tests for malaria

Recommendations for malaria diagnostic testing

• Suspected malaria should be considered a medical emergency ^{Footnote 5 Footnote 51 Footnote 52 Footnote 53}.
• Travellers to malaria-endemic areas should be advised to present themselves for medical attention, including laboratory assessment, as soon as possible but always within 24 hours of onset of an unexplained fever, both at any time while travelling and during the first year after returning. Travellers should always inform their health care provider of their travel history ^{Footnote 5}.
• Malaria should be suspected in any person presenting with a fever or history of fever and a history of travel to a malaria-endemic area ^{Footnote 2}.
• All suspected cases of malaria should have a parasitologic test to confirm the diagnosis.
• Blood should be examined immediately for malaria if it is suspected. If expertise in reading malaria smears is not available, diagnosis should involve the local use of an RDT and/or LAMP and then the rapid transfer of a blood sample to a reference laboratory (specimens with a negative LAMP do not need further workup). The result of the RDT, LAMP, or initial blood smear should be available within 2 hours of blood taking.
• If results are not available within 2 hours, the clinician should strongly consider empiric malaria treatment while awaiting results ^{Footnote 9}.
• If the initial smears are negative, and no alternate diagnosis is established, two additional smears should be taken and examined at 12 or 24 hour intervals ^{Footnote 54}.
• Before declaring the smears negative for malaria, someone experienced in smear analysis should examine a thin smear under oil immersion for 15–20 minutes (200–300 oil immersion fields at 100× magnification) and a thick smear for 5–10 minutes (200–300 oil immersion fields at 100× magnification) ^{Footnote 55 Footnote 56}. A negative LAMP is also sufficient to declare the sample negative for malaria.
• A laboratory should interpret the blood smear as positive or negative within 1–2 hours of receiving the blood, and should provide speciation within 12 hours, but if possible, immediately. Where available, PCR can also be used for speciation. Due to the increasing prevalence of HRP-2 mutant strains, absence of HRP-2 detection in the presence of a positive pan-malaria species result should not be interpreted as non-falciparum malaria without further confirmation. Quantification of parasitemia also should be available rapidly ^{Footnote 54 Footnote 55}. Parasitemia above 1% occurs mainly in P. falciparum and P. knowlesi infections. Thus if parasitemia in this range is suspected from examination of the smear, a formal count should be done and the clinician notified.
• RDTs or LAMP should be available in every clinical laboratory facility in Canada that might receive specimens for malaria diagnosis. RDTs or LAMP should be offered as the first line test, unless high quality malaria microscopy results can be made available within 2 hours of receipt of the specimens ^{Footnote 5}.
• Parasitemia is important for clinical management of P. falciparum and P. knowlesi. Some labs may find it to be more expeditious to report parasitemia for all specimens, rather than waiting for species confirmation ^{Footnote 57 Footnote 58}.
• RDTs and LAMP should not be used to assess response to therapy ^{Footnote 59 Footnote 60}.
• RDTs should not be routinely recommended for use by travellers ^{Footnote 5 Footnote 61 Footnote 62}.

Abbreviations: LAMP, loop-mediated isothermal amplification; PCR, polymerase chain reaction; RDT, rapid diagnostic test.

Acknowledgements

This chapter was prepared by: Libman M (Chair) and Plewes K (Malaria Sub-Committee Chair), and approved by CATMAT.

CATMAT would like to thank Lagacé-Wiens P and Desroches M (CATMAT Secretariat) for their contribution to updates to this chapter, and Boggild A and Payne E (CATMAT Secretariat) for their contribution to earlier versions of the chapter.

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