Hargobinder Kaur

RealAmp Loop-Mediated Isothermal Amplification as a Point-of-Care Test for Diagnosis of Malaria: Neither Too Close nor Too Far

TO THE EDITOR—Patel et al [1] investigated the usefulness of the real-time fluorescence loop-mediated isothermal amplification (LAMP) assay, RealAmp, as a point-of-care test in diagnosing malaria. In an accompanying editorial, Hsiang et al [2] highlighted some advantages and limitations of LAMP as a point-ofcare test. Molecular techniques are well known for their high sensitivity and specificity [3]. The same characteristics apply in the diagnosis of submicroscopic malaria by molecular techniques, compared with conventional microscopy or antigen-detection methods [4]. LAMP provides results rapidly and uses relatively inexpensive equipment for amplification, compared with conventional polymerase chain reaction (PCR) [5]. However, for validation of DNA extraction methods, it is essential to amplify housekeeping genes and to include positive and negative controls during each run. Because LAMP amplifies genomic DNA several fold, slight contamination during processing may result in false-positive findings, as highlighted byHsiang et al [2]. Thus, it is essential to analyze the results of RealAmp, along with the quality-control results, with caution. Another important factor that requires consideration is the DNA-extraction method of boil and spin, which has been used by Patel et al for onsite extraction of DNA. Briefly, this method requires boiling and centrifugation steps. Therefore, certain equipment and uninterrupted electricity supply are required if many samples have to be screened. Although the authors mentioned that equipment used for performing RealAmp can be run with the help of a rechargeable battery, in remote areas the supply of electricity may be insufficient for recharging the battery [6]. Moreover, whether a rechargeable battery can be used for long periods to power the equipment required for DNA extraction has not been investigated. For performing good-quality molecular work without contamination, separate workstations, along with sterile autoclaved tips and PCR tubes, are required. Before the use of PCR workstations, UV light exposure is required for decontamination. Thus, a regular electricity supply is essential to run an autoclave and for performing RealAmp, which may be the most limiting factor in remote areas. Innovations are required whereby DNA can be isolated by enzymes or reagents without sophisticated equipment, and LAMP can be performed in the same vial by adding reaction mixtures to it so that chances of contamination are minimized and amplification of DNA is achievable [7]. Further, automated systems can be developed in which all steps, starting with DNA extraction and isothermal amplification and continuing through the analysis of results, can be performed quickly, so that contamination can be avoided and the assay made more user friendly [8]. Thus, we are today neither too far nor too close to using LAMP as a point-ofcare test for the diagnosis of malaria.

Development of Visually Improved Loop Mediated Isothermal Amplification for the Diagnosis of Plasmodium vivax Malaria in a Tertiary Hospital in Chandigarh, North India

More than 80% of the global burden of the Plasmodium vivax is contributed by mainly three countries (India, Indonesia, and Pakistan). Reports from last decades have highlighted the occurrence of severe P. vivax malaria which was earlier considered to be benign. The recent trends of increasing P. vivax-associated morbidity and mortality emphasizes the need for early and accurate diagnosis of P. vivax malaria for the timely management of patients. Microscopy is considered a gold standard but needs experienced laboratory technologists. Over the last few years, Polymerase chain reaction (PCR) is being used as a highly sensitive and specific test but it requires expensive equipment which limits its use in the field. Therefore, in the present study, utility of visually improved loop-mediated isothermal amplification (LAMP) for the detection of P. vivax was evaluated targeting 18SrRNA gene in 145 microscopically confirmed P. vivax and 20 P. vivax negative patients. Sensitivity and specificity of LAMP was assessed with respect to microscopy and multiplex nested PCR (nPCR). Results of the LAMP assay was also correlated with rapid diagnostic test, multiplex nPCR and real-time PCR results. Overall, sensitivity and specificity of P. vivax-specific LAMP compared with microscopy were found to be 100% and 85%, respectively. Furthermore, detection limit for LAMP was found to be 0.8 copies/μL and it was also able to detect three complicated cases of P. vivax which were missed by microscopy. This study showed a LAMP assay to be a rapid and very sensitive method for the early diagnosis of both complicated and uncomplicated P. vivax malaria.

Genetic diversity of Plasmodium falciparum merozoite surface protein-1 (block 2), glutamate-rich protein and sexual stage antigen Pfs25 from Chandigarh, North India

To elucidate the genetic diversity of Plasmodium falciparum in residual transmission foci of northern India.

Distribution of mutations associated with antifolate and chloroquine resistance among imported Plasmodium vivax in the State of Qatar

Plasmodium vivax is the most prevalent parasite worldwide, escalating by spread of drug resistance. Currently, in Qatar, chloroquine (CQ) plus primaquine are recommended for the treatment of P. vivax malaria. The present study examined the prevalence of mutations in dihydrofolate reductase (dhfr), dihydropteroate synthase (dhps) genes and CQ resistance transporter (crt-o) genes, associated with sulphadoxine-pyrimethamine (SP) and chloroquine resistance, among imported P. vivax cases in Qatar. Blood samples were collected from patients positive for P. vivax and seeking medical treatment at Hamad General Hospital, Doha, during 2013-2016. The Sanger sequencing method was performed to examine the single nucleotide polymorphisms in Pvdhfr, Pvdhps, and Pvcrt-o genes. Of 314 examined P. vivax isolates, 247 (78.7%), 294 (93.6%) and 261 (83.1%) were successfully amplified and sequenced for Pvdhfr, Pvdhps, and Pvcrt-o, respectively. Overall, 53.8% (N = 133) carried mutant alleles (58R/117N) in Pvdhfr, whereas 77.2% (N = 227) and 90% (N = 235) isolates possessed wild type allele in Pvdhps and Pvcrt-o genes, respectively. In addition, a total of eleven distinct haplotypes were detected in Pvdhfr/Pvdhps genes. Interestingly, K10 insertion in the Pvcrt-o gene was observed only in patients originating from the Indian subcontinent. The results suggested that CQ remains an acceptable treatment regimen but further clinical data are required to assess the effectiveness of CQ and SP in Qatar to support the current national treatment guidelines. In addition, limited distribution of genetic polymorphisms associated with CQ and SP resistance observed in imported P. vivax infections, necessitates regular monitoring of drug resistant P. vivax malaria in Qatar.

Screening and identification of potential novel biomarker for diagnosis of complicated Plasmodium vivax malaria

BackgroundIn the recent years Plasmodium vivax has been reported to cause severe infections associated with mortality. Clinical evaluation has limited accuracy for the early identification of the patients progressing towards the fatal condition. Researchers have tried to identify the serum and the plasma-based indicators of the severe malaria. Discovery of MicroRNA (miRNA) has opened up an era of identification of early biomarkers for various infectious and non-infectious diseases. MicroRNAs (miRNA) are the small non-coding RNA molecules of length 19–24 nts and are responsible for the regulation of the majority of human gene expressions at post transcriptional level.MethodsWe identified the differentially expressed miRNAs by microarray and validated the selected miRNAs by qRT-PCR. We assessed the diagnostic potential of these up-regulated miRNAs for complicated P. vivax malaria. Futher, the bioinformtic analysis was performed to construct protein–protein and mRNA–miRNA networks to identify highly regulated miRNA.ResultsIn the present study, utility of miRNA as potential biomarker of complicated P. vivax malaria was explored. A total of 276 miRNAs were found to be differentially expressed by miRNA microarray and out of which 5 miRNAs (hsa-miR-7977, hsa-miR-28-3p, hsa-miR-378-5p, hsa-miR-194-5p and hsa-miR-3667-5p) were found to be significantly up-regulated in complicated P. vivax malaria patients using qRT-PCR. The diagnostic potential of these 5 miRNAs were found to be significant with sensitivity and specificity of 60–71% and 69–81% respectively and area under curve (AUC) of 0.7 (p < 0.05). Moreover, in silico analysis of the common targets of up-regulated miRNAs revealed UBA52 and hsa-miR-7977 as majorly regulated hubs in the PPI and mRNA–miRNA networks, suggesting their putative role in complicated P. vivax malaria.ConclusionmiR-7977 might act as a potential biomarker for differentiating complicated P. vivax malaria from uncomplicated type. The elevated levels of miR-7977 may have a role to play in the disease pathology through UBA52 or TGF-beta signalling pathway.

Molecular characterization of Plasmodium falciparum in Arunachal Pradesh from Northeast India based on merozoite surface protein 1 & glutamate-rich protein

Background & objectives: Northeast (NE) India is one of the high endemic regions for malaria with a preponderance of Plasmodium falciparum, resulting in high morbidity and mortality. The P. falciparum parasite of this region showed high polymorphism in drug-resistant molecular biomarkers. However, there is a paucity of information related to merozoite surface protein 1 (msp-1) and glutamate-rich protein (glurp) which have been extensively studied in various parts of the world. The present study was, therefore, aimed at investigating the genetic diversity of P. falciparum based on msp-1 and glurp in Arunachal Pradesh, a State in NE India. Methods: Two hundred and forty nine patients with fever were screened for malaria, of whom 75 were positive for P. falciparum. Blood samples were collected from each microscopically confirmed patient. The DNA was extracted; nested polymerase chain reaction and sequencing were performed to study the genetic diversity of msp-1 (block 2) and glurp. Results: The block 2 of msp-1 gene was found to be highly polymorphic, and overall allelic distribution showed that RO33 was the dominant allele (63%), followed by MAD20 (29%) and K1 (8%) alleles. However, an extensive diversity (9 alleles and 4 genotypes) and 6-10 repeat regions exclusively of R2 type were observed in glurp. Interpretation & conclusions: The P. falciparum population of NE India was diverse which might be responsible for higher plasticity leading to the survival of the parasite and in turn to the higher endemicity of falciparum malaria of this region.