Design and validation of multiplex polymerase chain reaction as a diagnostic tool for Plasmodium species.

BACKGROUND & OBJECTIVES: The highly sensitive method for a true understanding of malaria prevalence is of utmost importance for India's elimination strategy. The PCR reaction type with rapid detection, cost-effectiveness, and less workforce should be preferable. Multiplex PCR type accomplishes the present requirement by saving time and resources to find true surveillance data for malaria, especially in low-parasitemia/asymptomatic groups or populations. METHODS: The present study focuses on designing multiplex PCR (mPCR) to detect simultaneously Plasmodium genus (PAN) and two common Plasmodium species found in India. It is compared to standard nested PCR on 195 clinical samples to diagnose malaria. The mPCR was designed with a minimum number of primers, leading to less clogging and effective and enhanced detection. It contains one common reverse primer and three forward primers amplifying three targeted genes corresponding to P. falciparum, P. vivax, and Plasmodium genus. RESULTS: The sensitivity and specificity for mPCR were 94.06 and 95.74, respectively. The limit of detection for mPCR was 0.1 parasites/µl. The study has shown a ROC curve area for the mPCR of 0.949 for Plasmodium genus and P. falciparum and 0.897 for P. vivax with standard nPCR. INTERPRETATION & CONCLUSION: The mPCR is rapid in detecting species together, cost-effective, and requires fewer human resources than the standard nPCR. Therefore, the mPCR can be used as an alternative technique for the higher sensitive detection of the malaria parasite. It could also become a vital tool for determining malaria prevalence, facilitating the application of the most effective measures.

Quality assurance of malaria rapid diagnostic tests: An aid in malaria elimination.

Background & objectives: India targets malaria elimination by 2030 in a phased manner, so malaria's assured diagnosis is crucial. Introduction of rapid diagnostic kits in India in 2010 has revolutionized malaria surveillance. The storage temperature of rapid diagnostic tests (RDTs), kit components and handling in transportations impact the results of RDTs. Therefore, quality assurance (QA) is required before it reaches end-users. The Indian Council of Medical Research-National Institute of Malaria Research (ICMR-NIMR) has a World Health Organization (WHO) recognized lot-testing laboratory facility to assure the quality of RDTs. Methods: The ICMR-NIMR receives RDTs from different manufacturing companies as well as various agencies such as National and State Programmes and Central Medical Services Society. The WHO standard protocol is followed to conduct all the tests, including long-term and post-dispatch testing. Results: A total of 323 lots tested during January 2014-March 2021 were received from different agencies. Amongst them, 299 lots passed the quality of test and 24 failed. In long-term testing, 179 lots were tested and only nine failed. A total of 7741 RDTs were received from end-users for post-dispatch testing of which 7540 qualified the QA test with a score of 97.4 per cent. Interpretation & conclusions: RDTs received for quality testing showed compliance with QA evaluation of malaria RDTs based on the protocol recommended by the WHO. However, continuous monitoring of the quality of RDTs is required under QA programme. Quality-assured RDTs have a major role, especially in areas where low parasitaemia of parasites persists.

Comparative Assessment of Diagnostic Performance of Cytochrome Oxidase Multiplex PCR and 18S rRNA Nested PCR.

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

Prevalence of Asymptomatic Malaria Parasitemia in Odisha, India: A Challenge to Malaria Elimination.

The prevalence of malaria in India is decreasing, but it remains a major concern for public health administration. The role of submicroscopic malaria and asymptomatic malaria parasitemia and their persistence is being explored. A cross-sectional survey was conducted in the Kandhamal district of Odisha (India) during May-June 2017. Blood samples were collected from 1897 individuals for screening of asymptomatic parasitemia. Samples were screened using rapid diagnostic tests (RDTs) and examined microscopically for Plasmodium species. Approximately 30% of randomly selected samples (n = 586) were analyzed using real-time PCR (qPCR), and the genetic diversity of Plasmodium falciparum was analyzed. The prevalence of Plasmodium species among asymptomatic individuals detected using qPCR was 18%, which was significantly higher than that detected by microscopy examination (5.5%) or RDT (7.3%). Of these, 37% had submicroscopic malaria. The species-specific prevalence among asymptomatic malaria-positive cases for P. falciparum, Plasmodium vivax, and mixed infection (P. falciparum and P. vivax) by qPCR was 57%, 29%, and 14%, respectively. The multiplicity of infection was 1.6 and 1.2 for the merozoite surface protein-1 gene (msp1) and (msp2), respectively. Expected heterozygosity was 0.64 and 0.47 for msp1 and msp2, respectively. A significant proportion of the study population, 105/586 (18%), was found to be a reservoir for malaria infection, and identification of this group will help in the development of elimination strategies.