Chapter 6 - Malaria diagnosis: Canadian recommendations for the prevention and treatment of malaria
An Advisory Committee Statement (ACS) from the
Committee to Advise on Tropical Medicine and Travel (CATMAT)
Table of contents
- Rapid diagnostic tests
- Polymerase chain reaction
- Table 6.1: Comparison of diagnostic tests for malaria
- 6.2: Recommendations for malaria diagnostic testing
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.
Plasmodium falciparum malaria can be rapidly fatal, particularly in a non-immune host. It is the most urgent diagnosis to confirm or exclude in the febrile traveller who has been in a malaria-endemic zone. Although other signs and symptoms may be present in people with malaria, they are neither sensitive nor specific. For example, fever is frequently not cyclic, and splenomegaly is rarely present early in the course of P. falciparum malariaReference 1. Hence, clinical assessment, even by experts, cannot reliably confirm or exclude a diagnosis of malariaReference 2.
The large majority of travel-related malaria cases diagnosed in non-endemic countries present within several months of return from an endemic areaReference 3. However, some cases, including P. falciparum, will present after a longer period, that is, six months or more post-exposure. For this reason, the Committee to Advise on Tropical Medicine and Travel (CATMAT) 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.
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 recommendationsReference 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 attendant 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.
Confirmation or exclusion of a diagnosis of malaria depends upon laboratory examination of blood samples. 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 and for parasite speciation. Lack of experienced personnel may limit the accuracy of malaria diagnosis in laboratories in CanadaReference 5, whereas diagnosis in low-income countries is often hampered by problems with the quality of microscopes or stains and with supervision and quality control in the laboratoryReference 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 casesReference 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 1 to 2 hours of receiving a blood specimenReference 5. In very few cases, when the level of parasitemia is low, an initial smear may be falsely negative. Thus, one or two additional smears may be required every 12 to 24 hours to confirm or exclude the diagnosis, unless an alternate diagnosis can be firmly established. It is important to obtain repeat smears at regular intervals. There is no benefit in attempting to time blood sampling with the fever cycle, and this can potentially delay the diagnosisReference 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 making other decisions, for example, about hospitalization. Quantitating parasitemia for P. falciparum is also important in helping to determine the need for parenteral treatment or the need for admission to an intensive care unit. In addition, it is important for monitoring patient response to treatment.
Rapid diagnostic tests
Rapid diagnostic tests (RDTs) do not require microscopy or specialized laboratory skills and can play a valuable adjunctive role in diagnosing malariaReference 9. Varieties of RDTs are licensed by Health Canada for use in Canada and can be reviewed in the Medical Devices Active License ListingReference 10. RDTs are immunochromatographic assays that use monoclonal antibodies to capture malaria 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 reliabilityReference 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, parasite-specific lactate dehydrogenase (pLDH) or Plasmodium aldolase (also called panmalarial antigen). Lactate dehydrogenase-based tests may detect all species of malaria or may be specific for P. falciparum or P. vivax. Aldolase tests are panspecific and cannot differentiate between any of the types of malaria.
The World Health Organization lists 96 different tests that can be categorized into seven different typesReference 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 speciesReference 13. To date, tests specific for P. malariae and P. ovale are not availableReference 14, Reference 15. Recent studies have shown that P. knowlesi parasites possess both PfHRP-2 and pLDH, and therefore tests may falsely report P. falciparum or P. vivaxReference 16, Reference 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 due to lack of sensitivity for all non-falciparum species, a negative RDT does not exclude malariaReference 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 correctlyReference 19, Reference 20 and the rates of false-negative results are unacceptableReference 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, however; 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 RDTReference 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 polymerase chain reaction (PCR)Reference 9.
In general, RDTs are best at detecting P. falciparum, with sensitivities between 88% and 100% and specificities between 92% and 95%Reference 23. 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%)Reference 15. An earlier meta-analysis of RDTs in returned travellers found that tests based on PfHRP-2 were more accurate than those on pLDHReference 9. Sensitivity is decreased at parasite densities below 100/μL, with sensitivities of less than 70% at densities less than 50/μLReference 24. The sensitivity for the detection of P. vivax is inferior to that of P. falciparumReference 19, Reference 25. For P. vivax, the data are limited, but the threshold for satisfactory detection of parasitemia may be higher (> 1,000 parasites/μLReference 26. Rapid tests have only been positive in approximately 15% of patients with P. knowlesi on whom an RDT was used, and sensitivity of RDTs for P. vivax, P. malariae, and P. ovale in non-endemic areas is consistently less than 85%Reference 18.
RDTs are not recommended for assessing the response to antimalarial therapy. PfHRP-2 persists for prolonged periods after the asexual stage parasites (ie, those stages that are clinically relevant) have been cleared from blood by treatment, with 68% positivity at 7 days and 27% positivity at 28 days after initiation of therapyReference 24. Parasite aldolase-based tests also remain positive after clearance of asexual stage parasites and may remain positive even longer than PfHRP-2-based tests. In addition to slow clearance of parasite antigen leading to false positive RDT, isolated sexual stage parasitemia (i.e., gametocytemia), which is clinically irrelevant, and which can occur following successful treatment of malaria, may also lead to a positive RDT resultReference 27. The advantage of RDTs is that they are simple to use, require no equipment and can be performed by laboratory staff that 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 RDTsReference 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 antibodyReference 9, Reference 19. In addition, there are reports of false negative results for P. falciparum from multiple geographic regions due to strains genetically deficient in HRP-2Reference 28, Reference 29, Reference 30, Reference 31, Reference 32.
Occasionally, there may be cross-reaction between species. For example, the aldolase- or pLDH-based tests with falciparum and non-falciparum bands may give a positive reaction on both bands when only P. falciparum infection is present, making accurate diagnosis of mixed infections difficultReference 23. Geographic variation between P. falciparum strains could also affect test sensitivityReference 33.
Polymerase chain reaction
Although not practical for immediate care because of its limited availability, PCR is emerging as the gold standard for high sensitivity and specificity in speciation (Table 6.1). It is being increasingly used for quality control. PCR techniques (e.g. real-time PCR) that provide more rapid results are likely to become more available in the near futureReference 34, Reference 35, Reference 36.
|Approximate parasite density threshold,
per μL (%)
|Microscopy – thick films||50 (0.001)||Fair||Limited||No|
|Microscopy – thin films||> 100 (0.002)||Good||Limited||No|
|RDT||> 100 (0.002)||+/- (limited)||Good||No|
Abbreviations: PCR, polymerase chain reaction; RDT, rapid diagnostic test.
6.2: Recommendations for malaria diagnostic testing
- Suspected malaria should be considered a medical emergencyReference 5, Reference 37, Reference 38, Reference 39.
- 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 historyReference 5.
- Malaria should be suspected in any febrile person with a history of travel to a malaria-endemic area and a history of, or a finding of, feverReference 2.
- 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 then the rapid transfer of a blood sample to a reference laboratory. The result of the RDT 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 resultsReference 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 intervalsReference 40.
- 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)Reference 41, Reference 42.
- 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. Quantification of P falciparum also should be available rapidlyReference 40, Reference 41. Parasitemia above 1% is uncommon in non- P. falciparum malaria, and if parasitemia in this range is suspected from examination of the smear, a formal count should be done and the clinician notified.
- RDTs should be available in every clinical laboratory facility in Canada that might receive specimens for malaria diagnosis. RDT’s 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 specimensReference 5.
- RDT results (both positive and negative) must be verified by expert microscopy or PCR to determine the level of parasitemia and species identification. 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 confirmationReference 43, Reference 44.
- RDTs should not be used to assess response to therapyReference 45, Reference 46.
- RDTs should not be routinely recommended for use by travellersReference 5, Reference 47, Reference 48.
Abbreviations: PCR, polymerase chain reaction; RDT, rapid diagnostic test.
This chapter was prepared by: Libman M (lead) and Boggild A, and approved by CATMAT.
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