Abstract
BackgroundWith the documentation of cases of falciparum malaria negative by rapid diagnostic tests (RDT), though at low frequency from natural isolates in a small pocket of Odisha, it became absolutely necessary to investigate the status of HRP-2 based RDT throughout the state and in different seasons of the year.MethodsSuspected individuals were screened for malaria infection by microscopy and RDT in 25/30 districts of Odisha, India. Discrepancies in results were confirmed by PCR. False negative RDT samples for Plasmodium falciparum mono-infection were evaluated for detection of HRP2 antigen in ELISA and genotyped for pfhrp2, pfhrp3 and their flanking genes. Multiplicity of infection was ascertained based on msp1 and msp2 genotyping and parasitaemia level was determined by microscopy.ResultsOf the total 1058 patients suspected for malaria, 384 were microscopically confirmed for P. falciparum mono-infection and RDT failure was observed in 58 samples at varying proportion in different regions of the state. The failure in detection was due to undetectable level of HRP-2. Although most of these samples were screened during rainy season (45/345), significantly high proportion (9/17) of RDT negative samples were obtained during the summer compared to rainy season (P = 0.0002; OR = 7.5). PCR genotyping of pfhrp2 and pfhrp3 in RDT negative samples showed 38/58 (65.5) samples to be pfhrp2 negative and 24/58 (41.4) to be pfhrp3 negative including dual negative in 17/58 (29.3). Most of the RDT negative samples (39/58) were with single genotype infection and high proportions of pfhrp2 deletion (7/9) was observed in summer. No difference in parasitaemia level was observed between RDT positive and RDT negative patients.ConclusionHigh prevalence of parasites with pfhrp2 deletion including dual deletions (pfhrp2 and pfhrp3) is a serious cause of concern, as these patients could not be given a correct diagnosis and treatment. Therefore, HRP2-based RDT for diagnosing P. falciparum infection in Odisha is non-reliable and must be performed in addition to or replaced by other appropriate diagnostic tools for clinical management of the disease.
Highlights
With the documentation of cases of falciparum malaria negative by rapid diagnostic tests (RDT), though at low frequency from natural isolates in a small pocket of Odisha, it became absolutely necessary to investigate the status of HRP-2 based RDT throughout the state and in different seasons of the year
Of samples diagnosed positive for P. falciparum monoinfection by microscopy were found positive by RDT, whereas RDT failure was detected in 15.1% (58) of the cases
Most of the samples were screened and collected during rainy season (Table 1), significantly high proportion of (52.9%) RDT negative samples were obtained during the summer (P = 0.0002; OR = 7.5, 95% CI 2.75–20.45) compared to rainy season
Summary
With the documentation of cases of falciparum malaria negative by rapid diagnostic tests (RDT), though at low frequency from natural isolates in a small pocket of Odisha, it became absolutely necessary to investigate the status of HRP-2 based RDT throughout the state and in different seasons of the year. Clinical diagnosis being unreliable and non-specific, it required confirmation through parasitological diagnosis which involved identification of parasites by their direct observation in patient’s blood [such as microscopy and quantitative buffy coat (QBC) test], through identifying parasite nucleic acids by molecular methods [such as PCR, loopmediated isothermal amplification (LAMP) method, microarray] or parasite specific antigens/antibody by rapid diagnostic tests and other new strategies (such as mass spectrometry and flow cytometry). Each of these methods had their own limitations and many of these tests are non-amenable for point-of-care diagnosis, in resource-limited endemic regions. Both PfHRP2 and HRP3 share many structural similarities with signal peptide sequence located in both on exon 1, and exon 2 harbours histidine (H) and alanine (A) rich amino acid repeats [20]
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