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Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 541--546

High-sensitivity detection of human malaria parasites by the use of rapid diagnostic tests and nested polymerase chain reaction in burdened communities of North East India

Ramzan Ahmed, Kuldip Devnath, Deep Bhowmik, Indu Sharma 
 Department of Microbiology, Assam University, Silchar, Assam, India

Correspondence Address:
Dr. Indu Sharma
Department of Microbiology, Assam University, Silchar - 788 011, Assam
India

Abstract

Background and Objectives: The study aimed to evaluate the diagnostic performance of malaria through microscopy and rapid diagnostic test (RDT) analysis performed locally and the accuracy evaluated by nested polymerase chain reaction (PCR) for diagnosis of Plasmodium falciparum from hotspot regions of North East (NE) India. Materials and Methods: One thousand one hundred and seventy-three blood samples were collected for identification of P. falciparum infection using microscopy and RDT analysis. DNA was extracted from whole blood using QIAamp DNA blood mini kit, and nested PCR was performed to confirm P. falciparum for evaluating sensitivity and specificity from various epidemiological surveys and geographical areas of NE India. Results: Of 1173 symptomatic malaria suspected patients, 15.6% (183/1173) patients were diagnosed as malaria positive by RDT and 67.94% cases (53/78) with microscopy. Of 183 malaria-positive patients, 42.62% (78/183) were diagnosed with P. falciparum and 84.61% (66/78) further confirmed to be P. falciparum positive by nested PCR. High sensitivity (97.9%) and low specificity (2.03%) of the RDT and high sensitivity (99.1%) and low specificity (0.9%) in microscopy against nested PCR results was statistically significant (P < 0.05). Epidemiological comparisons expressed highest incidences in Manipur (51.11%) followed by Meghalaya (48.93%) and Assam (35.16%). Overall incidence rate among the genders was observed to be higher in males than in females. Conclusions: Our findings suggest that PCR, RDT and microscopy can potentially determine hotspots at moderate transmission intensities, but PCR testing has a diagnostic advantage as transmission intensity falls. Therefore, malaria control programs should consider PCR testing when the prevalence of infection is low.

How to cite this article:
Ahmed R, Devnath K, Bhowmik D, Sharma I. High-sensitivity detection of human malaria parasites by the use of rapid diagnostic tests and nested polymerase chain reaction in burdened communities of North East India.Indian J Med Microbiol 2018;36:541-546

How to cite this URL:
Ahmed R, Devnath K, Bhowmik D, Sharma I. High-sensitivity detection of human malaria parasites by the use of rapid diagnostic tests and nested polymerase chain reaction in burdened communities of North East India. Indian J Med Microbiol [serial online] 2018 [cited 2019 Aug 25 ];36:541-546
Available from: http://www.ijmm.org/text.asp?2018/36/4/541/254396

Full Text

 Introduction



Sadly, malaria is still today regarded as one of the highest killer and important parasitic diseases affecting tropical countries [1] with major challenges towards ease of diagnostic test performance and training of personnel.[2] Currently in the north eastern parts of India, malaria case detection especially in the remote areas depends heavily on microscopy method and till date is being regarded as one of the gold standard methods for malaria diagnosis towards identification of Plasmodium sp., with microscopic observations of thick and thin Giemsa-stained blood slides,[3] but on the other hand, rapid diagnostic tests (RDTs) have also practically proved a boon and the backbone for diagnosis of malaria infection in the developing as well as in highly remote areas where good microscopy facilities may be beyond the reach.[4] Since malaria eradication requires enhanced and regular surveillance along with control efforts, molecular tools, such as polymerase chain reaction (PCR), have proved to be highly sensitive in detecting low-density infections and determining the parasite species.[5] Although PCR limitations for its use as a routine diagnostic method in clinical settings are well known, it has still been used extensively in malaria surveillance since encouraging results have attracted increasing attention from the society and created awareness among the North East (NE) Indian population.

Thus keeping the view above, the present study was designed to adapt and evaluate this novel technique for the use in rapid laboratory-based detection of Plasmodium falciparum and to validate the sensitivity of this technique in comparison to that of conventional nested PCR [6],[7] with a potential for future applications in malaria epidemiological studies in hotspot regions of NE India.

 Materials and Methods



Study areas and sample collection

The present study was performed in the NE states of India (Assam, Meghalaya and Manipur) which have a predominance of P. falciparum and other mixed infections. Blood samples from 1173 symptomatic malaria patients were randomly collected from hospitals and private diagnostic centres and evaluated. Whole blood (K3 EDTA vial) and finger-prick blood samples were also collected from each enrolled patient on Whatman 3 mm filter (GE Healthcare) paper, air dried and individually placed in plastic bags for storage at appropriate temperature.

Rapid diagnostic tests

The Malaria antigen histidine-rich protein 2 (HRP2)/plasmodium lactate dehydrogenase (pLDH) Combo RDT test (Standard Q, SD Biosensor healthcare Pvt. Ltd., India) was used for diagnosing malaria infection containing the RDT pad monoclonal antibodies to HRP2 and pLDH antigen. The antigen–antibody complexes were captured here by monoclonal antibodies to the target antigens and immobilised on the test strip. The combo test detected the P. falciparum HRP2 (PfHRP2) antigen and a Plasmodium vivax pLDH antigen and interpretations were made according to the manufacturer's instructions.

Microscopy

Each sample was processed for preparation of thick and thin smears for detecting P. falciparum and stained with Giemsa stain following the WHO standards.[8] Thick blood smears prepared from peripheral blood were estimated using the standard value for the white blood cells (WBC) count (8000WBC/μl), and in thin blood smears, the parasitemia was estimated by noting the number of parasitised red blood cells (RBC) (not individual parasites) seen in 10,000 RBC and expressing the number of parasitised cells seen as a percentage. The stained thick and thin smears with Giemsa were screened for malaria parasites by microscopy with (×100) oil immersion. Smears were considered as negative when no parasite was observed in ×100 oil immersion fields in a thick blood film. In thick films, 200 oil immersion fields were evaluated before a sample was declared negative, and in thin films, the parasite count was established per 2000 RBC.

Nested polymerase chain reaction

One microliter of venous blood in K3 EDTA vial from adult patients and two–three drops of blood spots from every participant in case of minor were collected on Whatman 3 mm filter paper (GE Healthcare) and labelled with the participant's study code and date. Each filter paper was dried and stored individually in a zipper plastic bag for detection of P. falciparum by PCR assay with exclusion criteria of P. vivax and mixed infections from further molecular analysis.

DNA was extracted using QIAamp DNA blood mini kit (Qiagen, Germany) from filter paper blood spots and whole blood following the manufacturer's instructions. A slightly modified nested PCR amplification was performed as described previously based on 18s subunit rRNA gene.[9] For primary standard PCR reaction, 2 μL of genomic DNA was used in a 25 μL reaction volume with outer primers – rPLU1 and rPLU5. The primary PCR products have been used as DNA templates with the genus-specific primers – rPLU 3 and rPLU 4 in nested first PCR amplifications. PCR was performed under the same cycle conditions as mentioned above except for annealing temperature (64°C), which included an initial denaturation at 94°C for 5 min, 94°C for 1 min, 55°C for 2 min and 72°C for 2 min with a final extension for 5 min followed by 30 cycles. The nested first PCR product (2 μL) was used in second nested PCR for detection of P. falciparum species using species-specific primer – FAL 1 and FAL 2.

The amplified products were then separated in 1% and 2% agarose gels for primary and nested PCR, respectively, and stained with 2% ethidium bromide for visual detection of bands using ultraviolet transillumination.

Statistical analysis

Data were analysed using SPSS version 21.0 (IBM Corp., Armonk, New York, USA) for determining the sensitivity and specificity among the malaria-infected population. Considering the PCR standard in the diagnosis of malaria based on RDT and microscopic diagnosis of malaria, the performance of each diagnostic test method was calculated by means of sensitivity, specificity and accuracy to confirm the consistency of the results among the diagnostic tools. A 95% confidence interval (CI) was calculated and the value of P < 0.05 was considered statistically significant.

Ethical considerations

Ethical approval was obtained from Assam University, Silchar, Assam, India (Ref. no. IEC/AUS/06/2017). The study protocols were examined and approved by institutional ethical board members to conduct the present study along with informed and written consent from each patient involved with the study which was witnessed by a third party, which also maintained record and names of each participant as he/she enrolled with the study.

 Results



A total of 1173 symptomatic, malaria-suspected patients based on clinical presentation were screened for Plasmodium parasite in the study sites Assam, Meghalaya and Manipur, where 15.6% (183/1173) patients were diagnosed as malaria positive by RDT and 67.94% P. falciparum cases (53/78) with microscopy. Of 183 malaria-positive patients, 42.62% (78/183) were diagnosed with P. falciparum and 84.61% (66/78) further confirmed to be P. falciparum positive by nested PCR.

Performance of malaria rapid diagnostic test and microscopy in Assam, Meghalaya and Manipur

Of 1173 patients, a total of 868 patients were screened for malaria infection in Assam; 129 patients from Meghalaya and 176 patients from Manipur. It was observed that 91 patients from Assam were found to be infected with malaria followed by 47 patients in Meghalaya and 45 patients from Manipur. Further, 777 patients from Assam, 82 from Meghalaya and 131 patients from Manipur were excluded since they did not meet the inclusion criteria.

In Assam, 32 (35.16%) patients were diagnosed as P. falciparum positive by RDT diagnostic tool and 59 (64.83%) patients were diagnosed with mixed malaria species. Further results with microscopy revealed that only 21 (65.62%) patients were P. falciparum positive and 11 (34.37%) patients represented as P. falciparum negative. Of 868 (73.99%) total patients screened for malaria, 777 (89.51%) patients exhibited negativity towards P. falciparum; 23 (48.93%) patients were diagnosed as P. falciparum positive by RDT method and 24 (51.06%) patients were diagnosed with mixed malaria species. The results with microscopy revealed that only 17 (73.91%) patients were P. falciparum positive and 6 (26.08%) patients represented as P. falciparum negative, and from 129 (10.99%) total patients screened for malaria, 82 (63.56%) patients exhibited negativity towards P. falciparum whereas in Manipur, 23 (51.11%) patients were diagnosed as P. falciparum positive by RDT method and 22 (48.89%) patients were diagnosed with mixed malaria species. The results with microscopy revealed that only 15 (65.21%) patients were found to be P. falciparum positive and 8 (34.78%) patients were represented as P. falciparum negative, and from 176 (15.00%) total patients screened for malaria, 131 (74.43%) patients exhibited negativity towards P. falciparum.

Association of Plasmodium falciparum by polymerase chain reaction

The hotspot regions of NE India (Assam, Meghalaya and Manipur) showed that there was a strong positive correlation between the prevalence of parasitemia measured by PCR and prevalence of parasitemia measured by microscopy or RDT. Henceforth, the PCR assay confirmed 27 positive cases (84.37%) of P. falciparum malaria infection in Assam, 19 (82.60%) cases from Meghalaya and 20 (86.95%) from Manipur which had been earlier detected and confirmed by RDT method.

Furthermore, PCR assay of microscopy positive cases exhibited 19 (90.47%) of 21 P. falciparum cases and 8 negative cases which were later reconfirmed as PCR positive from Assam; 14 (82.35) cases out of 17 and 5 microscopy negative cases reconfirmed as positive cases by PCR from Meghalaya and 14 (93.33%) out of 15 microscopy positive samples from Manipur. Furthermore, 6 positive P. falciparum cases previously not diagnosed by microscopy were also confirmed.

To further test the accuracy of malaria RDT and microscopy, the samples were compared with PCR results. Among the samples with P. falciparum positive malarial infection by RDT and microscopy were confirmed by nested PCR and the accuracy of the tests were measured by receiver operating characteristic (ROC) analysis using SPSS software. ROC curve was drawn by a non-parametric method using SPSS software. The ROC curve obtained by plot at different cut-offs is shown in [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]. Therefore, the sensitivity and specificity of microscopy test when compared to PCR results in Assam was found to be 99.8% and 0.2% (area under the curve [AUC] = 0.999 with standard error [SE] = 0.001 and 95% CI from 0.997 to 1.000, P < 0.001) whereas RDT showed 99.4% and 0.6% (AUC = 0.997 with SE = 0.002 and 95% CI from 0.994 to 1.000, P < 0.001); 98.1% and 1.9% (AUC = 0.990 with SE = 0.007 and 95% CI from 0.977 to 1.000, P < 0.001) and RDT, 96.4% and 3.6% (AUC = 0.982 with SE = 0.011 and 95% CI from 0.961 to 1.000, P < 0.001) in Meghalaya and 99.4% and 0.6% (AUC = 0.997 with SE = 0.004 and 95% CI from 0.990 to 1.000, P < 0.001) and RDT was 98.1% and 1.9% (AUC = 0.990 with SE = 0.007 and 95% CI from 0.978 to 1.000, P < 0.001) in Manipur. Among the hotspot regions of NE India (Assam, Meghalaya and Manipur), epidemiological comparisons revealed the highest malaria incidences in Manipur (51.11%); Meghalaya (48.93%) and Assam (35.16%). The proportion of P. vivax and P. falciparum was also found to be almost at equal stage, but significant variations have been found to differ from place to place with seasonal influence. Furthermore, the overall malarial infection incidence rate among the genders was observed to be higher in males as compared to females. Assam exhibited 65.62% of the incidence rate of P. falciparum in males and 34.37% in females, followed by Meghalaya 76.26% in males and 21.73% females while in Manipur, 69.56% in males and 30.43% females were observed.{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}

 Discussion



The present study evaluated the accuracy of clinical diagnostic performance of malaria with respect to microscopy and RDT methods and further evaluation by nested PCR for diagnosis and confirmation of P. falciparum infections as gold standard. RDTs have been widely used for detection of different malaria-specific antigens prevalently P. falciparum-specific proteins; either PfHRP2 or P. falciparum lactate dehydrogenase (PfLDH) but they also have the ability to recognise both P. falciparum-specific and pan-specific antigens (aldolase [pALD] and pan-specific [pLDH]).[10] However, few studies have shown that due to the deletion of HRP2 gene, false-negative results may occur.[11],[12],[13],[14],[15],[16] Hence, the present study also recorded an overall exhibition of 12 (15.38%) false-positive malaria RDT cases along with negative PCR results for these 12 false-positive malaria cases. Thus, observations of such results could be due to malaria parasites expressing low level of target antigen or deleted pfhrp2 gene.[14],[16],[17],[18],[19],[20] Therefore, further research is required to determine the true prevalence of these mutations (deletion of pfhrp2) in high-prone epidemiological environments. Despite these limitations, HRP-2-based malaria RDTs was the preferred and observed to be the topmost choice, especially in the remote settings of NE India with limited microscopy facilities.[21]

Field evaluations of PfHRP2-based RDTs have reported high sensitivities in medium to high malaria transmission settings [22],[23] where parasite densities commonly exceed 200 parasite/μL but few studies have also shown reduction rate in sensitivity of RDTs for malaria detection in the present study; also, the accuracy of pfHRP2 antigen and microscopy test in detecting malaria infection against PCR assay among the symptomatic patients from the hotspot malarial zones of Assam, Meghalaya and Manipur (NE India) with varying intensities of transmission was observed. Further, RDT and microscopy performances were examined in relation to PCR and cases with false-positive HRP2 RDTs were re-examined using PCR assay to identify the P. falciparum species. Therefore, high sensitivity (97.9%) and low specificity (2.03%) of the RDT and high sensitivity (99.1%) and low specificity (0.9%) of microscopy against nested PCR results which was statistically significant (P < 0.05) for all the hotspot malarial zones of Assam, Meghalaya and Manipur were observed. Malaria prevalence was found to be moderate to low in Assam, Meghalaya and Manipur (35.16%, 48.93% and 51.11%) and significantly most of the case types were of adult patients. PCR as the reference diagnostic method performed better than microscopy and RDT. However, when the HRP2 RDTs and microscopy were considered together against PCR, improvement in sensitivity was observed in microscopy. In Assam, sensitivity and specificity for RDT antigen (Pf/Pv) test (Standard Q) against PCR was 99.4% and 0.6% and microscopy 99.8% and 0.2%; in Meghalaya, it was observed as 96.4% and 3.6% and microscopy test comparison to PCR was 98.1% and 1.9% and in Manipur, the sensitivity and specificity of RDT was found to be 98.1% and 1.9% and microscopy tests when compared to PCR was observed to be 99.4% and 0.6%, thus suggesting the accuracy of malaria diagnostic methods used herein. Comparisons with expert microscopy and PCR, the sensitivity value of malaria RDT was observed to be higher (≥95%) than the threshold percentage recommended by WHO [24] suggesting that such observations may be due to variant observer or differences with malaria species circulating at various regions. The nested PCR approach used in the present study proved to be simple and highly reproducible. We did not compare PCR with fresh blood versus blood spotted on filter paper; however, the dried blood spot technique proved to be far more practical, inexpensive, technically simple and once dried, the nucleic acids were found to be stable over a wide range of temperatures and over time.[25],[26] Our experience with the present study also confirms that RDT in conjunction with microscopy can improve the diagnosis of malaria especially in remote areas of NE India, and its use should be considered as most cost-effective in the regions that have been characterised by high-moderate intensity malaria transmission with particular focus and emphasising those regions where health services are deficient or almost negligible. The overall implications of this study are clinically relevant describing the change in the scenario of malarial epidemiology in the three hotspot zone of NE India accompanied along with other differential etiological agents and their molecular confirmations involved in the occurrence of malarial symptoms. Furthermore, variations in Plasmodium sp. and geographical distribution may be due to differences in genetic polymorphisms, underlying parasite drug resistance and host susceptibility, in addition to mosquito vector ecology and transmission seasonality;[27] hence, the presence and relevance of such factors must be addressed in future studies.

 Conclusions



RDTs may contribute to the treatment of symptomatic patients in different age group for malaria and non-malaria cases. This work revealed that a single RDT can be useful in the detection of HRP2 antigen for diagnosing malaria infection. Nested PCR method was more sensitive than RDTs and thus, malaria control programs increasingly need to adopt such control strategies even at low transmission intensities. Our findings therefore suggest that PCR, RDT and microscopy can potentially determine hotspots at moderate transmission intensities, but PCR testing has a diagnostic advantage as transmission intensity falls; thus, malaria control programs should consider PCR testing when the prevalence of infection is low.

Acknowledgements

We thank the study participants and field workers for their kind support and help in sampling.

Financial support and sponsorship

The authors are highly thankful to Assam University, Silchar, India for providing the necessary support in completion of the present work.

Conflicts of interest

There are no conflicts of interest.

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