ABSTRACT
Precise identification of Plasmodium species is critical in malaria control and elimination. Despite several shortcomings, microscopy and rapid diagnostic test (RDT) continue to be the leading diagnostic methods. Polymerase chain reaction (PCR) is the most sensitive method but its dependency on advanced laboratory and skilled workers limits its use. Here, we compared the diagnostic performance of microscopy, RDT, and PCR in clinically suspected patients from a high malaria burden state (Odisha) of India. The diagnostic performance (sensitivity, specificity, positive predictive value, and negative predictive value) of all three methods was compared using microscopy and PCR as the gold standard. PCR identified 323 (76.5 %) positive cases out of 422 samples, whereas microscopy and RDT identified only 272 (64.4 %) and 266 (63.0 %) positive cases, respectively. The sensitivity of RDT and microscopy for detecting malaria and P. falciparum cases was >80% compared to that by PCR. However, the sensitivity in identifying P. vivax (57.0 %) and a mixture of P. falciparum and P. vivax (18.0 %) was poor. We highlight application of PCR in malaria diagnosis and its benefits in reducing the transmission. This emphasizes the need for incorporation of molecular diagnostic approaches for effective elimination strategies.
KEYWORDS: Malaria, plasmodium, PCR, RDT, microscopy
Introduction
Malaria is one of the major public health problems in developing countries with global burden of 228 million cases and 405,000 deaths in 2018. Outside of Africa, India is a major contributor to this burden [1]. India reported 0.43 million malaria cases in 2018, with 47% cases of Plasmodium falciparum, and a vast majority of these cases from its rural areas mainly inhabited by tribal populations [2]. These communities live in hilly and dense forest areas where health care services, particularly disease diagnosis, are inadequate leading to poor reporting and disease management [3,4]. The World Health Organization (WHO) has recommended the T3 (Test, Treat, and Track) strategy to ensure accurate diagnosis of every suspected malaria case and treatment with appropriate antimalarial drugs to achieve global malaria elimination [5]. Microscopy and rapid diagnostic test (RDT) are used as the standard diagnostic tools in India. Microscopy is considered the gold standard in malaria diagnosis and is extensively used in district-level hospitals and community health centers (CHC) where adequate infrastructures exist. This method is less expensive and allows the identification of species and parasite density using thick and thin blood smears. The major limitations of microscopy are the need for experienced staff and reduced accuracy with low parasite count [6]. In contrast, RDT is considered a good alternative as it can be performed with untrained manpower and can be used at the community level. Usually, bivalent RDT based on histidine-rich protein 2 (HRP2) for P. falciparum and plasmodium lactate dehydrogenase (pLDH)/aldolase for P. vivax/PAN malaria is used for malaria diagnosis. Declining sensitivity of RDT with low parasitemia and its inability in differentiating mixed infection of P. ovale and P. malariae species has been reported [7]. False-negative results with HRP2 deletion parasites are yet another limitation of RDT [8]. To overcome these limitations, polymerase chain reaction (PCR) based on the detection of the 18S rRNA gene of the malaria parasite has been considered for laboratory diagnosis. PCR is the most sensitive method available that can detect even very low parasite count and allows accurate species identification. However, its application is limited to research laboratories and cannot be implemented in remote settings owing to its complex methodology, skilled manpower, reagent requirement and high risk of cross-contamination [9].
As India is moving toward malaria elimination, confirmatory parasitological diagnosis using highly sensitive tools such as PCR is essential to detect the clinical cases that may be missed by microscopy and RDT. The present study was carried out to identify the actual malaria burden by comparing three different diagnostic methods (microscopy, RDT, and PCR) among clinically suspected cases of malaria in a high burden state.
Materials and methods
Study site
The study was conducted at the Biswanathpur CHC of Kalahandi district, situated in the southwestern part of Odisha from April 2017 to January 2018. The annual parasite incidence in the district was 28.6 in 2016 [10]. The population of the Biswanathpur block is 23,395 (census 2011), and more than 50% is represented by scheduled castes and scheduled tribes (population living at the bottom of the social hierarchy as per government of India) involved in agriculture as the main occupation. The climatic condition of the district is generally hot with high humidity from March to August and an average rainfall of 1100 mm annually.
Sample size
The minimum sample size was determined to be 412 assuming 5 % discordance between different diagnostic methods (microscopy, RDT, and PCR) and 1.1 % absolute precision (marginal error) with an additional 5 % to account for sample loss.
Study procedure
The protocol of the study was approved by the Institutional Ethics committee of the National Institute of Research in Tribal Health, Jabalpur (Madhya Pradesh). Patients were enrolled after obtaining written informed consent from the patients or parents/legally acceptable representative of the children following ethical guidelines of the Indian Council of Medical Research, New Delhi. Pregnant women and patients with chronic diseases were excluded from the study. Clinically suspected (fever or malaria like illness) individuals were initially diagnosed with RDT (SD Bioline Malaria Antigen Pf/Pv Bio Standard Diagnostic Pvt. Ltd., India) and microscopy. RDT was performed and the result was analyzed as according to the manufacturer’s instructions. A thick and thin blood smear was prepared and stained with Giemsa. A total of 100 fields were examined under the oil immersion objective (100× magnification) prior to the slide being reported negative for malaria. Parasite density was calculated from thick blood smear as per WHO guideline [11] by calculating the number of asexual parasite × 8000/number of WBC counted. Quality control of microscopy (100% positive smears and 10% of negative smears) was done at ICMR-National Institute of Research in Tribal Health Jabalpur by a second expert blinded to the previous results. All malaria positive cases either by RDT or microscopy were treated as per national guidelines. Simultaneously, 1 ml venous blood samples were also collected and transported to the laboratory of the ICMR-National Institute of Research in Tribal Health, Jabalpur, for molecular analysis. Genomic DNA was isolated from 200 µL of blood sample using an eco-prep® blood DNA extraction kit (Genetix Biotech Asia Pvt. Ltd) according to the manufacturer’s instructions. Species-specific nested PCR targeting 18S rRNA gene was performed as described by Krishna et al [12]. Microscopy and RDT were performed by an independent technician and PCR by a research assistant to avoid biases.
Statistical analysis
All the data were entered in Microsoft Excel 2010 worksheet and exported in R v3.5.0 for Windows (R foundation for statistical computing) for statistical analysis. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) with a 95% confidence interval were calculated for all malaria species in case of P. falciparum, P. vivax, and mixed (PF + PV) infection detected by microscopy, RDT, and PCR. Microscopy and PCR were considered a gold standard for analysis. Diagnostic performance of microscopy, RDT against PCR, and RDT, PCR against microscopy was compared by receiver operating characteristics analysis.
Results
A total of 425 clinically suspected cases were enrolled in the study and screened for malaria. Analysis was performed with 422 samples since three samples were excluded from the study owing to inadequate sample quantity. Out of the total samples, 55.4% were males and 44.5% were females, and their mean age was 24 ± 17 years.
Out of the 422 samples, 76.5% (323/422) were positive for malaria by PCR, 64.4% (272/422) by microscopy, 63.0% (266/422) by RDT, and 62.79% (265/422) by all three diagnostic methods. The discordant results in the different diagnostic methods showed that 1.6% (7/422) were positive by microscopy and PCR but negative by RDT. Furthermore, 12% (51/422) cases were identified as positive by PCR but not by microscopy and RDT (Figure 1). Of the 51 cases detected only by PCR, 78.4% (40/51), and 5.9% (3/51) were found to be P. falciparum and P. vivax, respectively. P. malariae and P. ovale were also found in 1.9% (1/51) and 3.9% (2/51) samples, respectively. Moreover, mixed infections of PF + PM and PF + PV were detected in 1.9% (1/51) and 7.8% (4/51) samples, respectively (Figure 2). All the RDT-positive cases were confirmed by PCR and microscopy except one, which was negative by both PCR and microscopy.
Figure 1.
Venn diagram showing comparative malaria prevalence by microscopy, RDT, and PCR
Figure 2.
Flow diagram showing malaria diagnosis using microscopy, RDT, and PCR in Kalahandi District of Odisha
Considering microscopy as a gold standard, the sensitivity of RDT was 97.4% and specificity 99.3%.However, in the cases of P. vivax and mixed infection (PF + PV), the sensitivity and specificity of RDT was 100%. Furthermore, the sensitivity of RDT and microscopy against PCR as the gold standard was found to be 82% and 84.2% for all malaria positive and P. falciparum cases, respectively, while it was considerably low at 57.0% for P. vivax and 18.0% for mixed infection (Table 1). Area under curve (AUC) of microscopy and RDT against the PCR method for malaria positive cases was 92% (90%–94%) and 91% (88%–93%), respectively Similarly, AUC of PCR and RDT against microscopy for malaria positivity was 83% (79%–87%) and 98% (97%–99%), respectively (Table 1)
Table 1.
Comparative diagnostic performance of microscopy, RDT, and PCR
| Microscopy as reference method |
PCR as reference method |
||||
|---|---|---|---|---|---|
| RDT | PCR | RDT | Microscopy | ||
| Sensitivity (95% CI) |
total positive | 97.4% (94.8%–99%) | 100% | 82% (77.4%–86%) | 84.2% (79.8–88%) |
| PF | 97.3% (94.5%–98.9%) | 90.1% (85.8%–93.4%) | 82.9% (78%–87.2%) | 85.5% (80.8%–89.4%) | |
| PV | 100% | 100% | 57.1% (18.4%–90.1%) | 57.1% (18.4%–90.1%) | |
| mix | 100% | 100% | 18.2% (6.9%–35.5%) | 18.2% (6.9%–35.5%) | |
| Specificity (95% CI) |
total positive | 99.3% (96.3%–100%) | 66% (57.8%–75.5%) | 98.9% (94.5%–99.9%) | 100% |
| PF | 99.4% (96.6%–100%) | 75% (67.5%–81.5%) | 81.5% (74.2%–87.4%) | 82.2% (75%–88%) | |
| PV | 100% | 99.3% (97.9%–99.9%) | 100% | 100% | |
| mix | 100% | 93.5% (90.7%–95.7%) | 100% | 100% | |
| PPV (95% CI) |
total positive | 99.6% (97.4%–100%) | 84.2% (81%–86.9%) | 99.6% (97.4%–99.9%) | 100% |
| PF | 99.6% (97.3%–100%) | 85.5% (81.8%–88.6%) | 89.4% (85.7%–92.3%) | 90% (86.5%–92.8%) | |
| PV | 100% | 57.1% (30.2%–80.5%) | 100% | 100% | |
| mix | 100% | 18.2% (13.4%–24.2%) | 100% | 100% | |
| NPV (95% CI) |
total positive | 95.5% (91%–97.8%) | 100% | 62.8% (57.2%–68.1%) | 66% (60.1%–71.5%) |
| PF | 95.8% (91.6%–97.9%) | 82.2% (76%–87%) | 71.7% (65.8%–76.8%) | 75% (69.1%–80.1%) | |
| PV | 100% | 100% | 99.3% (98.3%–99.7%) | 99.3% (98.3%–99.7%) | |
| mix | 100% | 100% | 93.5% (92.5%–94.4%) | 93.5% (92.5%–94.4%) | |
| AUC (95% CI) |
total positive | 98% (97%–99%) | 83% (79%–87%) | 91% (88%–93%) | 92% (90%–94%) |
| PF | 98% (97%–99%) | 83% (79%–86%) | 82% (78%–86%) | 84% (80%–88%) | |
| PV | 100% | 99% (99%–100%) | 79% (59%–98%) | 79% (59%–98%) | |
| mix | 100% | 97% (96%–98%) | 59% (52%–66%) | 59% (52%–66%) | |
Out of 272 microscopy positive samples, association of parasite density with malaria positivity and species identification by RDT and PCR was analyzed for 268 samples; four P. vivax samples were excluded owing to absence of parasite count. With parasitemia lower than 100 parasites/µL (the lowest parasite density detected by microscopy was 32 parasite/µL) RDT failed to detect six P. falciparum cases, which were positive by microscopy. In contrast, PCR identified all microscopy positive cases including PF + PV + PM + PO (n = 2), PF + PV (n = 2), and PF + PM (n = 1) that microscopy identified as P. falciparum only. At >1000 parasites/µL, RDT identified all microscopy positive cases including one mixed (PF+PV). On the other hand, PCR detected four additional mixed (PF+PV) that was identified P. falciparum by microscopy. The sensitivity of RDT was further improved with an increase in parasite density but in the case of mixed infection, RDT was ineffective in detecting PF + PV cases as compared to that by PCR even at a high parasite count (Table 2).
Table 2.
Parasite density verses malaria positivity and species identification by RDT and PCR
| |
|
Total positive |
Mono PF |
Mixed infection |
|||
|---|---|---|---|---|---|---|---|
| Parasite density |
Microscopy Positive |
RDT Positive %, (n) |
PCR Positive %, (n) |
RDT Positive %, (n) |
PCR Positive %, (n) |
RDT Positive %, (n) |
PCR Positive %, (n) |
| 1–100 | 64 | 91(58/64) | 100(64/64) | 91(58/64) | 92.2(59/64) | No cases | 3.1(2/64) PF+PV, 1.5(1/64) PF+PM and 3.1(2/64) PF+PV+PM+PO |
| 101–1000 | 49 | 98 (48/49) | 100(49/49) | 98(48/49) | 91.8(45/49) | No cases | 8.1(4/49) PF+PV |
| 1001–5000 | 50 | 100(50/50) | 100(50/50) | 98(49/50) | 90(45/50) | 2(1/50) | 10(5/50) PF+PV |
| 5001–10,000 | 26 | 100(26/26) | 100(26/26) | 92.30(24/26) | 84.61(22/26) | 7.69(2/26) | 15.38(4/26) PF+PV |
| >10,000 | 79 | 100(79/79) | 100(79/79) | 96.20(76/79) | 82.27(65/79) | 3.79(3/79) | 15.18(12/79) PF+PV,1.26(1/79) PF+PO and 1.26(1/79) PF+PM |
| Total | 268 | 97.4(261/268) | 100(268/268) | 95.1(255/268) | 88.1(236/268) | 2.2(6/268) | 11.9(32/268) |
Discussion
Although Odisha represents only 3.3% of the population of India, it is one of the most affected malaria-endemic states in India because of various factors [13,14].
Despite substantial decline in malaria cases in 2018, Odisha is still a major contributor to malaria in the country. Large parts of the state are covered with hilly dense forests resulting in ineffective malaria control [15]. In such settings, false-negative cases by microscopy and RDT pose a major challenge, affecting transmission and disease outcomes. Therefore, accurate diagnosis is critical for malaria control and elimination.
Our results revealed that PCR identified an additional 12 % (51/422) and 13.7 % (58/422) cases which were missed by microscopy and RDT, respectively. A possible reason could be low parasite count [16,17] and inadequately trained technicians in the case of microscopy [6]. In the present study, we detected 78 % (40/51) of the infection missed by microscopy were due to P. falciparum. Similar observation was also reported by Berzosa et al. [18] who identified >90 % false-negative cases with P. falciparum in microscopy (n = 326/335) and RDT (n = 122/128) in comparison to that by PCR. PCR identified three additional P. vivax cases that were negative by microscopy and RDT due to comparatively low density of parasite in P. vivax infection as compared to that of P. falciparum in settings where both species occur [19]. We also observed seven P. falciparum cases that were positive in both microscopy and PCR but negative in RDT. This may be owing to pfhrp2 gene deletion leading to false-negative result with RDT [8,20].
The sensitivity and specificity of microscopy and RDT in detecting positive cases were >80% using PCR as a reference method. Although the specificity of both microscopy and RDT in detecting mixed and P. vivax infections was 100%. However, in the case of P. vivax, their sensitivities were very low (57%). These findings are concordant with the results of other studies [21–24]. Additionally, we also observed PF+PV mix infection by PCR which was diagnosed as PF by microscopy and these missed cases may increase the possibility of drug resistance [25]. Two cases of quadruple mixed infections (PF+PV+PM+PO) were observed which also diagnosed as P. falciparum by microscopy and findings reported by Krishna et al (7) established that high transmission area with all four parasite are present in the similar geographical region.
The present study is also in-line with other studies where sensitivity increases with an increase in parasite density [26,27]. Although both microscopy and RDT were found to be effective test methods (AUC = 80%–90%) in detecting malaria and P. falciparum cases, while competence in detecting P. vivax was relatively low (AUC = 79%) with PCR as gold standard.
The present study highlights the lack of sensitivity in microscopy and RDT in places where mixed infection and non-falciparum species exist. As India is already on its way to eliminate malaria, it is essential to use the molecular methods to achieve and sustain the elimination particularly in the clinically suspected malaria cases which were negative by microscopy and/or RDT. The approximate cost of molecular test would be 200 INR (3$), if the large volume of sample is diagnosed routinely the recurring cost may also be reduced. Considering an example of tuberculosis control and elimination program of India, the cartridge-based nucleic acid amplification test (molecular diagnostic test) has been implemented in all districts with plans to expand further to benefit peripheral centers. Furthermore, the indigenously developed TrueNat tests are planned to be set up in 350 PHCs for tuberculosis diagnosis [28]. A similar strategy for molecular detection is needed in rural setting for malaria diagnosis which will not only impact on case management but also prevent the transmission and help in achieving the elimination goal in the stipulated time frame. It is worth to mention that regular quality check system should be established to maintain the proficiency of the laboratory for molecular test. Although there are limitations of molecular method in terms of cost and low accessibility in resource limited settings but there are various portable tools like Gazelle and other low-cost tools (approximate 60–70 INR or 1$) which fulfill the purpose of detecting low-level parasite in malaria elimination settings [29,30]. Implementing the molecular detection test at district/CHC system may have hurdled (trained human resource, infrastructure and supplies in terms of consumables) during the initial phase but the current situation force to equip our health system with modern sensitive diagnostic laboratory for disease control and elimination.
Conclusion
Microscopy and RDT remain the preferable methods in the field settings but lacks sensitivity in detecting non-falciparum and mixed infection cases. In addition, microscopy and RDT were unable to detect high number of P. falciparum cases that were identified by PCR. The present study highlights the need for the adoption of nucleic acid-based molecular tools to accurately identify malaria in inaccessible settings to accelerate malaria elimination.
Acknowledgments
We would like to thank Tata Trust for funding to this study and Indian Council of medical research for approving the protocol of the study. We are grateful to all the study participants and their relative for providing information. We thank all staff of study site particularly medical officer for their support. The manuscript has been approved by the publication screening committee of ICMR-NIRTH Jabalpur
Funding Statement
This research was part of one of the funded study by Tata Trust.
Authors’ contributions
AA, PS, LK perform experiments, PS and LK performed data collection, AA, MPS, AKV, AS and PKB prepare manuscript, MPS perform data analysis, PKB, AKV and AS create study design and PKB, AD, AKV and AS involved in review of manuscript.
Disclosure statement
Author declare no conflict of interest
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