Comparative evaluation of traditional and molecular diagnostic methods for malaria: An analysis of performance

Abstract

Purpose:

As we edge closer to the eradication of malaria, several methods for detecting Plasmodium species have been developed, including peripheral blood smear examination (PBS), rapid diagnostic tests (RDTs), serological evaluations, fluorescent microscopy, polymerase chain reactions (PCRs), fluorescent in situ hybridization, and flow cytometry. The suitability of these tools for routine diagnosis requires evaluation, considering both their diagnostic accuracy and cost-effectiveness.

Materials and Methods:

Our study compared four diagnostic techniques for malaria: PBS, quantitative buffy coat (QBC), RDT, and PCR. We used PCR as the benchmark standard and statistically assessed the performance of PBS, QBC, and RDT against PCR in detecting malaria. Adopting a prospective observational approach, we collected blood samples from 117 patients exhibiting the symptoms suggestive of malaria.

Results:

The findings from our study showed that PBS had a positivity rate of 93.4%, with a 95% confidence interval (CI) of 0.881–0.987, indicating reliable results for a similar population. The QBC assay demonstrated an elevated positivity rate of 96.7% with a solid 95% CI of 0.930–1.000. Although the RDT had a slightly lower rate of 92.4%, it still delivered dependable results, presenting a significant 95% CI of 0.868–0.980, ensuring a robust diagnostic performance compared to PCR.

Conclusion:

PCR is a reliable test when the identification of the specific species is inconclusive. Conversely, the commonly used PBS occasionally overlooks positive malaria cases due to the specialized skills needed for accurate reading. The cost-effective RDT is feasible for field operations without the need for expert knowledge. However, it fails to differentiate between old and new infections. Meanwhile, the QBC test, known for its sensitivity and speed, can be consistently employed for malaria diagnosis in a tertiary care settings.

INTRODUCTION

Malaria remains a significant global health challenge, posing a major burden on public health systems, particularly in the endemic countries. As of 2019, the World Health Organization estimated 229 million cases and 409,000 deaths due to malaria worldwide.[1] In 2020, the South-East Asia region significantly contributed to the global burden of malaria, coming in as the second-largest contributor worldwide after the African region. The region reported approximately 5 million cases and incurred a substantial loss of approximately 9000 lives due to the disease.[2] Accurate and timely diagnosis is of paramount importance for effective disease management, control, and eventual elimination of malaria. Traditional diagnostic methods for malaria, such as peripheral blood smears (PBSs), have several limitations, including variable sensitivity and specificity, and dependence on the expertise of the microscopist.[3] The development of alternative diagnostic techniques, such as quantitative buffy coat (QBC), rapid diagnostic tests (RDTs), and polymerase chain reaction (PCR), has shown the potential to address these limitations, offering improved sensitivity and specificity. PCR in particular has been demonstrated to be a highly sensitive and specific diagnostic tool, especially in cases of low parasitemia.[4,5] However, the practicality of implementing these diagnostic tests in various settings, including hospital and field conditions, remains a crucial consideration.

Considering advancements in malaria diagnostic tests and the overarching goal of malaria elimination, the present study aims to compare the performance of three malaria diagnostic tests – QBC, PBS, and RDT with PCR, which served as the gold standard for comparison. In addition, this study has assessed the applicability of these diagnostic tests in both hospital and field settings by drawing the comparisons with similar published studies, thereby contributing to the ongoing efforts toward disease elimination in India. Through this comparative analysis, the study seeks to identify the most effective diagnostic methods suitable for diverse settings, facilitating the implementation of targeted malaria elimination strategies.

MATERIALS AND METHODS

Study design and study population

In this prospective, observational study, conducted at a tertiary health-care facility in the southern region of Karnataka, India, the primary objective was to evaluate and compare the efficacy of various diagnostic modalities, including PCR, QBC, RDTs, and PBS, in the accurate diagnosis of malaria. For 12 months, a random sample of 117 individuals presenting with clinical signs and symptoms indicative of malaria, such as fever, chills, rigor, headache, muscle aches, and jaundice were taken into this study. Among these, 92 patients with confirmed positive malaria status through nested PCR were included in the study, while an additional 25 patients exhibiting clinical symptoms of malaria but yielding negative PCR results served as the negative control group. Ethical approval for the study was granted by the institutional ethical committee under reference number IEC 514/2015. Blood specimens were collected through venipuncture using ethylenediaminetetraaceticacid vacutainers as the part of the standard diagnostic process, and subsequent analyses were conducted to determine the diagnostic accuracy of the various malaria detection methods.

Examination of peripheral blood smear

Thick and thin blood films were prepared and air-dried, with thin films subjected to methanol fixation. The smears were subsequently stained with Leishman’s stain and examined using oil immersion under light microscopy.[6] In the absence of any parasitic forms suggestive of Plasmodium species after examining at least 100 consecutive oil immersion fields, thick films were deemed malaria negative.

Quantitative buffy coat analysis

This technique relies on the stratification of blood components subjected to high-speed centrifugation, conducted using a Q.B.C.T.M. Capillary blood tube (Lilac Medicare, Thane, India). A 60 μl blood sample was drawn into a capillary tube precoated with acridine orange and centrifuged at 12,000 rpm for 5 min. The buffy coat was subsequently examined for parasitic forms indicative of the malaria parasite.

Rapid diagnostic test

The malaria Ag Pf/Pv kit (SD Bioline, Gurgaon, India) was employed for the RDT, utilizing an immunochromatographic cassette format to detect Plasmodium falciparum and Plasmodium vivax antigens. The test identified the histidine-rich protein 2 of P. falciparum and Plasmodium lactate dehydrogenase of P. vivax. A test result was considered negative if no test line emerged after 15 min of incubation.

Polymerase chain reaction analysis

Molecular diagnosis was performed using a fluorescence-based real-time PCR malaria differentiation kit (Fast-track diagnostics, Luxembourg), which is standardized to detect four Plasmodium species: P. vivax, P. falciparum, Plasmodium malariae, and Plasmodium ovale. DNA was extracted from whole blood samples using a Qiagen blood mini kit and subjected to amplification reactions performed with an ABI 7500 thermocycler. Following the manufacturer’s instructions, a cycle threshold of <36 was deemed positive for the detection of malaria species. PCR was taken as a gold standard for this study.

Statistical analysis

IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. (Armonk, NY: IBM Corp.USA) was used for the analysis. Comprehensive analysis of performance metrics was done, which included sensitivity, specificity, positive and negative predictive values (NPVs), diagnostic accuracy, and positive and negative likelihood ratios, to gauge the efficacy of each diagnostic method. We established a statistical significance threshold at P < 0.05, considering any results below this value to hold statistical significance.

RESULTS

A cohort of 117 patients who exhibited clinical symptoms suggestive of malaria or were suspected cases of malaria relapse underwent concurrent testing using four diagnostic methods: PCR, QBC, PBS, and RDT. Employing PCR as the gold standard, 92 cases were PCR confirmed positives, and 25 were PCR confirmed negative, these negative cases were considered a negative control group, as shown in Table 1. Among this negative control group, 10 (40%) samples tested positive for the dengue antigen, 4 (16%) were leptospirosis positive, 8 (32%) showed significant bacteremia, 2 (8%) were diagnosed with scrub typhus, and 1 (4%) presented with a urinary tract infection, indicated by the presence of Escherichia coli in the urine culture. Breaking down the PCR-positive cohort further, 40 out of 92 (42.5%) patients were diagnosed with P. falciparum, whereas 52 (57.5%) had P. vivax. Out of 117 patients, malaria was not detected in 6, 3, and 7 cases, respectively, by the routinely used diagnostic tests in our laboratory, namely the PBS, the QBC, and the RDT. These patients fall into the subpatent category, meaning their infections were not detectable by standard diagnostic tests. Passive surveillance was done in all cases, which involved diagnosing these cases as they presented them with suspected malaria, such as fever, chills, headache, anemia, muscle aches, and jaundice.

Table 1.

Total number of patients tested with each method, the number of positive and negative results confirmed by polymerase chain reactions, and the number of false positives and false negatives for each method

Total diagnostic test	Total tested	Positive (PCR confirmed)	Negative (PCR confirmed)	False positives	False negatives
PBS	117	86	25	0	6
QBC	117	89	23	2	3
RDT	117	85	22	3	7

PBS: Peripheral blood smear, QBC: Quantitative buffy coat, RDT: Rapid diagnostic test, PCR: Polymerase chain reactions

Table 1 presents the results of three diagnostic tests for malaria: PBS, QBC, and RDT. The results are compared to PCR testing, which is considered the gold standard for malaria diagnosis in this study.

Looking at the PBS results, 86 were correctly identified as having malaria (PCR confirmed), and 25 were correctly identified as not having malaria (PCR confirmed). There were no false positives (cases where PBS indicated malaria, but PCR did not), but there were 6 false negatives (cases where PBS did not indicate malaria but PCR did). The initial PBS test may have missed some cases of malaria, due to low-parasite density in the blood that was below the detection limit of the microscopy. The PCR test was able to detect the presence of the malaria parasite in these patients. After the PCR confirmation, the patients were retested with PBS. It is possible that by this time, the parasite density in their blood had increased to a level that could be detected by microscopy, resulting in a positive PBS result. So possibly, this highlights the importance of using sensitive diagnostic methods like PCR, especially in cases where the parasite density might be low and difficult to detect with microscopy.

Polymerase chain reaction-negative but quantitative buffy coat-positive and rapid diagnostic test-positive cases

There were two patients in the PCR-negative group who were QBC-positive, and three patients who were RDT-positive. These cases were retested by PCR, and the results were found to be again negative by PCR, it would suggest that the initial QBC-positive and RDT-positive results were likely to be false-positive cases. These false positive results could be due to various reasons such as cross-reactivity with other substances in the tests, technical errors, or issues with the specific test kits used. In terms of clinical outcomes, these patients were either asymptomatic or had mild symptoms that resolved on their own, it might suggest that they did not have a current malaria infection.

As shown in Table 2, PBS demonstrated a positive result percentage of 93.4% with a 95% confidence interval (CI) of 0.881–0.987, signifying reliable results if repeated in a similar population. The QBC assay reported a slightly higher positive percentage of 96.7%, indicating a strong confidence in its ability to detect malaria with a 95% CI of 0.930–1.000. RDT, while exhibiting a slightly lower percentage of 92.4%, still held a robust 95% CI of 0.868–0.980, suggesting reliable diagnostic performance. Collectively, these tests exhibit a high efficacy in diagnosing malaria in patients with fever-like symptoms.

Table 2.

The rate of positive test outcomes among patients presenting with fever-like symptoms, broken down by each diagnostic test used

Diagnostic test	Positive percentage (95% CI)
PBS	93.4 (0.881–0.987)
QBC	96.7 (0.930–1.000)
RDT	92.4 (0.868–0.980)

PBS: Peripheral blood smear, QBC: Quantitative buffy coat, RDT: Rapid diagnostic test, CI: Confidence interval

Diagnostic accuracies of quantitative buffy coat, peripheral blood smear, and rapid diagnostic test as compared to polymerase chain reaction

In our study, we evaluated the diagnostic accuracy of three testing methods for malaria: PBS, QBC assay, and RDT [Table 3]. When compared with PCR, which was used as the gold standard. QBC assay outperformed both PBS and RDT in terms of sensitivity of 96.7% as compared to PBS’s 93.4% and RDT’s 92.4%. This means that the QBC was more accurate in identifying true positive cases of malaria. On the other hand, PBS demonstrated superior specificity with a perfect score of 100%. This means that PBS was able to accurately identify all the individuals who did not have malaria. The QBC assay had a specificity of 92%, and RDT followed with 88%, implying that PBS is more efficient in reducing false-positive cases. In terms of positive predictive value (PPV), which reflects the probability that subjects with a positive screening test truly have the disease, PBS and QBC showed high levels of accuracy with scores of 100% and 97.8%, respectively. RDT also demonstrated a high PPV of 96.6%. Finally, for the NPV, which is the probability that subjects with a negative screening test truly did not have the disease, QBC held the highest score of 88.5%, followed by PBS at 80.6%, and RDT at 75.9%. This indicates that among the three methods, QBC is the most reliable in correctly identifying individuals who are truly free of the disease when the test result is negative. In short, while PBS shows excellent specificity and PPV, QBC holds the upper hand in sensitivity and NPV. RDT, while slightly less precise, still exhibits strong performance, making all three techniques effective and valuable tools in malaria diagnosis. Each method has its strengths and could be utilized effectively depending on the specific context and resources available.

Table 3.

Diagnostic accuracy test results of quantitative buffy coat, rapid diagnostic test, and peripheral blood smear in comparison to polymerase chain reaction

Diagnostic test	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
PBS	93.4	100	100	80.6
QBC	96.7	92	97.8	88.5
RDT	92.4	88	96.6	75.9

PBS: Peripheral blood smear, QBC: Quantitative buffy coat, RDT: Rapid diagnostic test, PPV: Positive predictive value, NPV: Negative predictive value

We also evaluated and compared the diagnostic performance of the RDT with other published studies [Table 4]. The results demonstrated a sensitivity of 92.4%, a specificity of 88%, a PPV of 96.6%, and a NPV of 75.9%. When comparing our findings to previous studies, our sensitivity and specificity were quite similar to those reported by Djibrine et al. (92.3% and 81.2%, respectively) and Tadesse et al. (99.5% and 98% for P. falciparum and 92.6% and 100% for non-P. falciparum). Our PPV was higher than the 85% reported by Djibrine et al., but slightly lower than the 98.4% (P. falciparum) and 100% (non-P. falciparum) reported by Tadesse et al. As for NPV, our study’s result was lower than those reported in all other studies.[7,8,9,10,11,12] Despite some variations, the SD Bioline RDT demonstrated strong diagnostic performance across all studies.

Table 4.

Diagnostic performance of rapid diagnostic tests for various studies

Parameter	Our study (n=117)	Djibrine et al. (n=436) (Djallé et al., 2014)	Tadesse et al. (n=374) (P. falciparum) (Tadesse et al., 2016)	Tadesse et al. (n=374) (non-P. falciparum) (Tadesse et al., 2016)	Tseroni et al. (n=955) (Tseroni et al., 2015)
Sensitivity (%)	92.4	92.3	99.5	92.6	97.3
Specificity (%)	88	81.2	98	100	99.4
PPV (%)	96.6	85	98.4	100	86.1
NPV (%)	75.9	90.1	99	94.4	99.9

P. falciparum: Plasmodium falciparum, PPV: Positive predictive value, NPV: Negative predictive value

DISCUSSION

PBS examination has been a traditional diagnostic method since Laveran’s time and, over a century later, it is still considered the gold standard for detecting malaria.[5] It is broad acceptance stems from its affordability and its capacity to estimate parasite density while identifying the species and stage of the malaria parasite. However, it requires considerable expertise and is labor intensive. Previous studies have demonstrated that this method struggles to detect malaria parasites reliably at low parasitemia (5–10 parasites/μl),[6] which was also observed in our study when six patients with malaria were missed by PBS examination but later diagnosed by PCR. In line with Kilian et al.’s findings, microscopy tends to underestimate mixed P. falciparum and P. vivax infections, a shortfall also observed in our study where mixed infection cases were not identified by PBS examination but were detected by QBC and PCR.[6,7] Therefore, simpler tests such as RDT are invaluable in resource-limited areas where such expertise may be lacking.[6,7,8]

The QBC has proven to be a promising tool for diagnosing malaria, as illustrated in our study where the sensitivity and specificity were 96.7% and 92%, respectively. In comparison, Parija et al., reported a sensitivity of 78.9% and a specificity of 98%.[9] This disparity may stem from interobserver variability during the QBC examination. Despite being a subjective test prone to interobserver variation, it is high sensitivity renders this test a standout among other diagnostic methods. Therefore, in health facilities like ours, equipped with trained staff and resources, QBC. serves as an excellent screening tool.

RDTs play a crucial role in diagnosing malaria in areas with limited resources. As an objective test, it can also prove useful when speciation is inconclusive by PBS examination and QBC. Various studies have evaluated the diagnostic performance of RDT. Using the Malaria Ag Pf/Pv kit (SD Bioline, Gurgaon, Haryana, India), as shown in Table 1. With robust diagnostic performance, this kit has proven to be an invaluable diagnostic tool.[9,10,12]

While molecular techniques such as PCR are the most sensitive tools for malaria detection, cost constraints make their routine use questionable in resource-limited settings. However, in fully equipped laboratories and tertiary care health-care facilities, PCR could be instrumental in diagnosing the disease when other tests fall short. Owing to its high accuracy in detecting mixed infections, PCR is useful when species identification by other tests is inconclusive. Studies like those conducted by Echeverry et al. suggest that PCR could be an excellent alternative for accurately detecting Plasmodium species.[8] Similarly, Alam et al.’s study suggests that it could serve as an alternative gold standard in reference settings, a notion aligned with our experience.[13] Looking ahead, PCR could be further standardized to detect emerging species such as Plasmodium knowlesi, given its high fatality rate and the need for special attention.

Study limitations

Despite the study’s limitations in terms of sample size, all three tests demonstrated high sensitivity compared to PCR. The choice of test largely depends on resource availability and local malaria endemicity, with each playing a distinct role in the goal of eradicating malaria. Furthermore, it is crucial to highlight the need for appropriate malaria treatment and control measures. Equally important is fostering understanding and awareness about the critical choice of the diagnostic test used for detecting the parasite.

CONCLUSION

The QBC is a promising diagnostic tool considering its performance metrics. However, both RDT and PBS, as objective tests, play a vital role in identifying species, particularly in instances of mixed infections. PCR stands out as an excellent tool for detecting submicroscopic malaria, especially in regions with low-malaria transmission. Therefore, PCR could potentially serve as an alternative gold standard test in future.

Ethical statement

Ethical approval was obtained from the institutional ethics committee before commencing the study (Ref: IEC 514/2015). Written informed consent was obtained from all participants.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.