Date Published: June 14, 2019
Publisher: Public Library of Science
Author(s): Amreen Ahmad, Anil Kumar Verma, Sri Krishna, Anjana Sharma, Neeru Singh, Praveen Kumar Bharti, Luzia Helena Carvalho.
Accurate and timely diagnosis is very critical for management, control and elimination of the malaria. Malaria rapid diagnostic tests (RDTs) have improved the diagnosis and management of malaria in remote areas, community and places where microscopy is not available for diagnosis. According to WHO report 2018, Plasmodium falciparum malaria constitutes more than 50% of malaria cases in India. Most of the RDTs used for diagnosis of falciparum malaria today employ HRP2 as a target antigen. However, low density parasitemia and deletion of hrp-2 gene in P. falciparum leads to false negative results and necessitates the development of alternative/ new or improved RDT for malaria diagnosis. We have analysed the genetic diversity and homology modelling of Pfgdh (glutamate dehydrogenase), ldh (lactate dehydrogenase) and aldolase genes in P. falciparum isolates from the eight endemic states of India to assess their potential as antigen for RDT development. We observed negligible sequence diversity in Pfgdh in comparison to the low level of diversity in ldh and aldolase gene. No structural or functional changes were observed in modelling studies and all three genes were under negative purifying selection pressure. The highly conserved nature of pfgdh gene suggests that GDH could be a potential target molecule for Pan/Pf diagnostic test for malaria.
Malaria imposes heavy health and socioeconomic burden on tropical and sub-tropical regions of the world with an estimated 219 million cases and 435 000 deaths in 2017 . India contributed about 85% of total malaria burden in the South East Asia Region . Majority of malaria cases and deaths in India are reported from rural areas of the state with dominant tribal population. These include Odisha, Chhattisgarh, Jharkhand, Madhya Pradesh, Maharashtra, West Bengal, Karnataka, Assam Gujarat and North- Eastern (NE) states . In these states, malaria endemic districts are located in hilly, densely forested, remote, inaccessible areas and are often marred by inadequate health care facilities. Lackadaisical health care seeking behaviour and socio-economic backwardness add further salt to injury . The WHO has set the goal of malaria elimination by 2030 . However, this aim can only be achieved when all the indigenous cases of malaria are diagnosed correctly and treated properly through the available diagnostic tools like microscopy, Rapid Diagnostic Test (RDT) and Polymerase Chain Reaction (PCR). Microscopy is considered ideal for malaria diagnosis but it has several limitations including requirement of skilled microscopist for interpretation of results and poor sensitivity in detecting low-level parasitaemia . With RDT, it is possible to diagnose malaria instantly in the field, rural and poor health care settings where microscopy is not available and clinicians have to make quick decision for case management. The ease of handling and prompt diagnosis has made malaria RDTs more popular. Since, its introduction, the sales of malaria RDTs has grown tremendously with distribution of 1.92 billion RDT globally during 2010–2017. About 276 million RDT were sold in 2017 alone in the world. Due to high prevalence of malaria, the WHO Africa region was on the biggest consumer of RDT (223 million) in 2017 . The sales and use of RDT has increased from 10.6 million to 15.2 million with the total use of 118.1 million RDT units for malaria diagnosis during 2010–2017 in India, [1, 5].
A total of 514 microscopy and PCR positive samples (which were available in sufficient quantity) for P. falciparum were selected for present study from a previous PfHRP-2 study . The sizes of pfgdh, pfldh and Pfaldolase amplified gene products were 1243bp, 924bp and 921bp respectively.
Diagnosis is critical for achieving the goal of malaria elimination set by the government of India by 2030. Rapid diagnostic tests (RDTs) are the major diagnostic tool particularly used in field and community surveys in malaria endemic countries with under-equipped health care infrastructures. The WHO has issued recommendations about the choice of RDTs, however selection of right RDT remains difficult for most users in endemic countries because of variation in targeted antigen in local parasitic population [34, 35]. One of the most important, but least studied of these factors is genetic variability of the target antigens of malaria parasite. [36, 37].