In a case series of imported malaria in the late 1990s in Canada, up to 92% of patients who presented to physicians without expertise in tropical medicine experienced delays in diagnosis due to physician failure to consider malaria as a diagnosis on initial presentation, laboratory error in recognition and species identification, or administration of incorrect therapy to treat the parasite(s) identified [9]. In this study, the majority of community laboratories did not perform malaria smears on an urgent basis, nor did they routinely report species identification or percent parasitaemia [9]. Overall, these laboratories had significant reporting delays, misdiagnoses and incorrect species identification [9]. Additionally, proficiency testing in the U.S. in the last decade has revealed especially poor identification of non-falciparum species of malaria [10]. A decline in number of cases reported that include species identification has also been seen in the malaria surveillance data reported to the Centers for Disease Control and Prevention (CDC) (Figure ​(Figure1)1) [3-7].

The Clinical and Laboratory Standards Institute's (CLSI) recommendations for laboratories performing malaria diagnostic testing include [11]:

1. Make diagnostic testing available 24 hours a day, seven days a week.

2. Prepare at least three thick and thin smears.

3. Use Giemsa stain for definitive diagnosis. It allows proper visualization of stippling (Schűffner's dots) in Plasmodium vivax and Plasmodium ovale and can play a crucial role in identification of these species. Although parasites are visible by Wright's and Wright-Giemsa stains, these stains do not allow visualization of stippling.

4. Examine at least 300 fields using the 100× oil immersion objective.

5. Report microscopy results immediately to the requesting physician or ward.

6. Examine at least 10 fields on the thin film to determine percent parasitaemia; many more fields should be examined for patients from the U.S. who typically have lower parasitaemia (CDC recommends counting parasitized red blood cells (RBCs) among 500-2,000 RBCs on the thin smear [12]).

A preliminary report of positive or negative should be available within four hours; percent parasitaemia should be reported within six hours and species identification within 24 hours.

A recent retrospective review by Edson et al analysing the proficiency of U.S. laboratories to identify malaria parasites reported a failure rate of 11.2% for identification of Plasmodium falciparum [10]. Although this means that more than one in 10 specimens were not correctly identified, it is encouraging compared with Canadian and British laboratory failure rates of 27% and 21% respectively [13,14]. However, given the potential for significant morbidity and mortality associated with delayed or incorrect diagnosis of P. falciparum, improvement is needed.

In this study, the majority of laboratories surveyed reported using both thick and thin smears; very few respondents reported the use of thin smears alone, an inferior technique. It is important that both thick and thin smears be evaluated in the examination of blood specimens for malaria. Thick smears are useful for detecting parasites, while thin smears are useful for species identification and quantification of parasites.

A few respondents reported that diagnostic testing was available only during the eight- to twelve-hour work day with no access to on-call personnel during the off-hours. Malaria is a potentially life threatening disease: therefore, it is recommended that all blood films be read immediately. CLSI recommends that 'any laboratory providing the expertise to identify malarial parasites should do so on a 24-hour basis, seven days a week' [11]. Therefore qualified personnel who can perform this testing should be on-call during off-hours.

Very few respondents reported availability of serologic testing in their laboratories; several other respondents reported its availability as a send-out test. Serologic testing is used only for retrospective diagnosis of past malaria infection [15] and is not recommended for diagnosis of acute infection. Anti-malarial antibodies are produced one to two weeks after initial infection and persist for three to six months after parasite clearance. Malaria RDTs are being used more frequently over the last several years, primarily in malaria-endemic areas[16]. Microscopy remains the gold standard, and in the U.S. is the primary and in some cases the only diagnostic tool used; as evidenced by the small number of respondents who reported having access to RDTs in their laboratories. RDTs can be beneficial in laboratories lacking personnel proficient in the microscopic diagnosis of malaria, as they allow for a rapid diagnosis of malaria, which can then be confirmed by microscopy or PCR. RDTs utilize immunochromatographic methods to detect malarial antigens present in peripheral blood to provide a quick result as to whether or not a patient is infected with malaria. Certain RDTs are able to give limited information about species (i.e. to indicate that at least P. falciparum is present) but none are able to provide quantification of parasitaemia. There are many brands and types of RDTs worldwide, but only one is FDA-approved for diagnostic use in the U.S.: BinaxNOW Malaria^® (Inverness Medical, Princeton, NJ). This test targets both the histidine-rich protein II (HRPII) antigen specific to P. falciparum and a malarial aldolase common to all human malaria species; it is therefore able to provide a diagnosis of malaria as well as determine specifically if P. falciparum is present[17]. However, all results must be confirmed by microscopy under the product's current FDA approval.

The detection of parasites in non-immune individuals who may be symptomatic at very low parasite densities is an additional barrier; unless enough fields are counted, these specimens may be mistakenly read as negative. In such cases it is imperative that repeat blood smears be performed. Standard guidelines recommend that a total of three specimens be obtained before declaring a patient does not have malaria. It is important to note that ensuring that an adequate number of specimens are sent is the responsibility of the physician; however, the laboratory may assist the physician with a reminder that three specimens must be sent to guarantee an accurate result.

Any laboratory with the capacity to perform a complete blood count with manual differential has the tools necessary to make a diagnosis of malaria and to report the percent parasitaemia (Additional file 2). All haematology technicians should have the basic training to identify a parasite on peripheral blood smear. Parasite speciation requires additional training and may therefore require sending the specimen to a reference laboratory. CDC provides diagnostic assistance, most rapidly (within 12-24 hours) through telediagnosis, where digital photographs and basic epidemiological data are sent to on-call personnel (See Additional file 2 for contact information*). Additionally, online diagnostic instructions are available at CDC's parasitology diagnostic website, DPDx [18] (See references for website address). For laboratories without on-site malaria diagnostic testing, consideration should be given to implementing RDTs, with confirmation via off-site testing.

Accurate identification of the infecting species has significant implications for treatment, as patients with P. vivax and P. ovale infections require therapy with primaquine to prevent relapses from latent hypnozoites in the liver. Accurate species identification is also important for detecting changes in the epidemiology of malaria among travellers. Laboratories should ensure that a definitive species-level diagnosis is made; PCR is a useful tool for definitively determining the Plasmodium species present. In addition, use of PCR should minimize the number of false negative and false positive results. PCR is more sensitive than microscopy for detecting mixed infections [19,20], as these are easily missed on a blood smear if morphologic features of both species are not visualized.

Although a high percentage of respondents reported compliance with each individual CLSI guideline, only very few representatives surveyed reported compliance with all CLSI guidelines. The purpose of these guidelines is to standardize practices across laboratories, as well as to recommend practices which will ultimately lead to the best outcomes through rapid identification and reporting of positive malaria smears. While species identification is critical to ensure that appropriate definitive treatment is given, providing a rapid presumptive diagnosis of malaria is most important to ensure prompt treatment is given. Therefore, while ideally all CLSI guidelines should be adhered to, those aimed at improving the rapidity and accuracy of initial reporting, such as counting an adequate number of fields and an adequate number of smears, are the most important.

Figure ​Figure66 outlines a useful algorithm for laboratories receiving specimens for malaria diagnostic testing. A preliminary report of positive or negative should be available within 4 hours; percent parasitaemia should be reported within 6 hours and species identification within 24 hours. This may be more difficult in laboratories where tests are sent out; laboratory supervisors should facilitate rapid retrieval of these results to ensure physicians have the information necessary to optimally manage the patient. Otherwise, they should consider making RDTs available to facilitate the initial diagnosis. If this is not possible, specimens should be referred to a laboratory where malaria diagnostic testing can be performed in a timely fashion. Patient care is a collaborative effort which includes many members of the medical team. In addition to the vital role of the laboratory staff, it is paramount for physicians to obtain a complete history, including travel history, and keep malaria high on the differential diagnosis of patients presenting with fever. As physicians it is our responsibility to ascertain the availability of diagnostic tests in our institution's laboratories and, if they are lacking, advocate for more timely access to better malaria testing.