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Sensitivity and Specificity of Malaria Screening
June 2018 - Vol. 7 No. 5 - Page #16

Malaria is a life-threatening disease that infected approximately 216 million people worldwide in 2016 resulting in 445,000 deaths, the majority of which were children in sub-Saharan Africa.1 The disease is diagnosed through microscopic identification of parasites within red blood cells on Giemsa-stained thick and thin blood films. In addition to this methodology, PCR molecular testing detects plasmodium species DNA (which may be residual nucleic acid present after effective treatment or be representative of viable intact parasites), in the specimen as an adjunct to conventional microscopy.

There are five species of plasmodium known to produce disease in humans: falciparum, vivax, ovale, malariae, and knowlesi. Differentiation of plasmodium species is important for both prognosis and therapy direction, yet in the United States, there are a relatively few (1,500) cases of malaria diagnosed each year. Thus, the considerable expertise required for microscopic diagnosis and speciation is difficult to achieve given the low volume of cases any pathologist or laboratory professional is likely to encounter outside of a high-volume center.

Compare In-house and Outsourced Results

Between January 1, 2013 and August 31, 2015, there were 142 malaria smears performed at Banner University Medical Center - Tucson (BUMCT) in Tucson, Arizona. Of those, 82 were referred out for confirmation and speciation by microscopy and PCR to a large reference laboratory.2 This was done in accordance with the current BUMCT laboratory protocol, which specifies that all positive smears be referred out for confirmation and speciation, while negative smears need only be sent for outside consultation if there is strong clinical suspicion of the disease.

The results of the 82 blood smears for malaria were compared to confirmatory testing by expert microscopy and PCR to assess the sensitivity and specificity of the in-house malaria smear relative to the gold standard of both expert microscopic review and PCR. Analysis found the in-house sensitivity to be 94% and the specificity to be 98% (the positive predictive value of the screen is 0.94 and the negative predictive value is 0.99). These findings have clinically important implications for clinical pathology laboratory management in the context of value-based testing.

Identify Potential Pitfalls

Potential pitfalls of in-house identification of these organisms on films are twofold: First, given the paltry number of these tests performed each year at our institution, it is difficult for physicians to maintain the diagnostic skills and experiential proficiency necessary for confirmation and speciation of the plasmodium parasite. Second, our laboratory does not perform a simple Giemsa stain. The lab uses a dual Wright-Giemsa stain, which is the standard stain for peripheral blood smears but can obfuscate identification and speciation of the parasite. Regardless, all positive or highly clinically suspicious smears are sent out for confirmation at outside laboratories where the special Giemsa stain is performed to aid in speciation. With these challenges in mind, a project was established to more accurately determine the sensitivity and specificity of BUMCT microscopy results compared to the gold standard of speciation by malaria specialists at the reference laboratory.

Methods and Results

In order to have a large enough database, the project reviewed sample results from January 2010 to August 2015. The results were analyzed for comparison of the in-house smear results with those obtained from the reference laboratory, including PCR (see Table 1).

Of the 82 smears sent for confirmation and speciation, there were 15 true positives, 65 true negatives, one false negative, and one false positive. Relative to the gold standard (confirmation and speciation by smear and PCR), the BUMCT test sensitivity was found to be 94% with specificity at 98%. The positive predictive value of the screen is 0.94 and the negative predictive value is 0.99. Of the true positives, there were seven cases of P. falciparum, four cases of P. ovale, one case of P. vivax, and one case of P. malariae species. There also were two cases of Babesia that were confirmed by the same method (see Table 2).

Discrepant analysis of the one false positive and one false negative was performed by chart review of the case history. The false positive involved a 2-year-old child of Ethiopian descent who was later diagnosed with adenovirus and giardiasis. The false negative occurred in a 33-year-old man with recent travel history to Kenya and Rwanda who presented with fever, chills, body aches, malaise, nausea, and vomiting. In this case, PCR identified DNA of P. falciparum in the patient’s sample, but the confirmatory lab reassured that the organisms were undetectable by smear.

Value of In-house Testing

Current protocols notwithstanding, appreciating the rigor of the in-house malaria screen is important because it informs the clinician in the first hours to days after presentation of symptoms. Prompt treatment during this time frame is crucial for the patient’s well-being. The CDC reports that delay in treatment is a leading cause of death in malaria patients in the US, but does not recommend empirical/presumptive treatment with antimalarial medication.1 If the screen result is positive, an antimalarial regimen can be initiated while confirmation and speciation is pending. Rapid diagnostic tests are available that can confirm the presence of the parasite, but are highly dependent on parasite density in the sample and can neither determine species nor quantify the parasitemia, both of which are crucial for effective treatment.

Regarding the malaria screen performed at BUMCT, a specificity of 98% and negative predictive value of 0.99 supports the proposition that a negative smear result is a reliable result and automatically sending out for confirmation by smear and PCR may not be warranted, even if the clinical suspicion is considered to be high. As an institution, this is meaningful in the context of value-based testing, with attention to minimizing superfluous testing and addressing the increasing cost of patient care. While it may seem presumptuous to report a definitive negative result without confirmation, especially given the paucity of smears evaluated annually, the results of this study maintain that the in-house screen is as rigorous as the outside consultation and rapid diagnostic testing in terms of ability to detect parasites in red blood cells.

Financial Impact

Per institution inquiry, confirmatory microscopy costs $68.32 and PCR costs $142.02 for a total send out cost of $210.34 per specimen. The total cost was $13,882.44 for the 66 negative smears that were sent out for confirmatory testing, which arguably neither provided clinically significant information, nor informed or expedited appropriate medical management.

Considering the single false negative identified by this analysis, a legitimate concern may be raised that without confirmatory testing by PCR the patient who had a falsely negative smear would have been misidentified and therefore potentially mistreated. Fortunately, there is an additional safety feature inherent in the clinico-pathologic diagnosis of malaria as prescribed by the CDC, which is repeat testing every 12-24 hours for a total of three sets, with the diagnosis of malaria only ruled out once all three results are deemed negative.1 In the case of the patient with a single falsely negative blood smear, the subsequent samples collected were found to be positive and resulted before confirmatory PCR results were received. The patient was started on anti-malarial medication without unnecessary delay.


The finding of a sensitivity of 94% and a positive predictive value of 0.94 are the less rigorous of the two parameters calculated in this study, meaning the laboratory is more likely to report a false positive than a false negative. Because current protocol at BUMCT mandates that positive screens be confirmed by PCR in all cases, there is a safety net guarding against this type of error. However, outside confirmation and PCR can take several days to result, so it is important to demonstrate the quality of the in-house screen and communicate this information to the clinician, who then can be confident in initiating treatment when appropriate.

Considering laboratory management as it pertains to patient care, it would be advisable to identify opportunities to increase the sensitivity of the screen and provide greater value to both the clinician and patient. Because P. falciparum and P. knowlesi infections have the potential to rapidly progress to severe illness and death, prompt identification of these species and initiation of appropriate treatment is of utmost importance, clinically. Given the turnaround time for confirmation and PCR results, training directed toward identifying these species specifically and reporting their presence preliminarily may help direct urgent patient care before outside confirmation of a positive smear is returned.

Sidebar. Use of Wright-Giemsa Stain

The use of the Wright-Giemsa (W-G) stain is an issue of resource conservation and avoiding duplicate processing. The W-G stain is standardly used for all peripheral smears and serves multiple purposes, only one of which is identifying potential parasites. While it is disadvantageous for this purpose, the overall utility of the W-G stain is what makes it so commonly used. Using a W-G stain, presence of malarial parasites in red blood cells can be detected, but speciation is difficult. At this point, speciation is primarily obtained using PCR rather than microscopy, which makes speciation from the smear less important diagnostically. Most labs in the US see very few positive malaria smears, so rather than performing the special Giemsa stain in house and not knowing how to interpret it necessitating a send-out anyway, the lab chooses not to offer the special stain as any abnormal smear (still easily recognized using a W-G stain) will be sent out for PCR and microscopic evaluation by an expert in malarial parasitology, who will perform the special Giemsa stain.


  1. US Centers for Disease Control and Prevention. Parasites; Malaria. CDC Malaria Program website. Accessed 5/16/18.
  2. Mayo Clinic Laboratories Test ID: LCMAL page:

Bagambhrini Gerace, MD, received her BS from the University of Arizona in neuroscience and cognitive science with a minor in fine arts and music. She completed her MD with distinction from the University of Arizona, during which she spent a year as a fellow in the clinical and anatomic pathology department of Banner University Medical Center in Tucson. She is now a resident in family and community medicine at Penn State Hershey Medical Center.


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