Malaria is a vector-borne infectious disease, caused by five different species of the genus Plasmodium, and is endemic to many tropical and sub-tropical countries of the globe. At present, malaria ...diagnosis at the primary health care level in India is conducted by either microscopy or rapid diagnostic test (RDT). In recent years, molecular diagnosis (by PCR assay), has emerged as the most sensitive method for malaria diagnosis. India is highly endemic to malaria and shoulders the burden of two major malaria parasites, Plasmodium falciparum and P. vivax. Previous studies using PCR diagnostic assay had unraveled several interesting facts on distribution of malaria parasites in India. However, these studies had several limitations from small sample size to limited geographical areas of sampling. In order to mitigate these limitations, we have collected finger-prick blood samples from 2,333 malaria symptomatic individuals in nine states from 11 geographic locations, covering almost the entire malaria endemic regions of India and performed all the three diagnostic tests (microscopy, RDT and PCR assay) and also have conducted comparative assessment on the performance of the three diagnostic tests. Since PCR assay turned out to be highly sensitive (827 malaria positive cases) among the three types of tests, we have utilized data from PCR diagnostic assay for analyses and inferences. The results indicate varied distributional prevalence of P. vivax and P. falciparum according to locations in India, and also the mixed species infection due to these two species. The proportion of P. falciparum to P. vivax was found to be 49:51, and percentage of mixed species infections due to these two parasites was found to be 13% of total infections. Considering India is set for malaria elimination by 2030, the present malaria epidemiological information is of high importance.
Since 2010, malaria rapid diagnostic tests (RDTs) are widely used to detect malaria. The Indian Council of Medical Research-National Institute of Malaria Research performed lot testing (LT) according ...to WHO procedures since 2016. Lot testing is performed to evaluate the lot-to-lot variation in performance of malaria RDTs. Four sets of positive quality control (QC) panels for P. falciparum (Pf) and P. vivax (Pv) and 10 negative panels tested RDTs. RDTs were reported as pass, failed, or deferred on the basis of WHO criteria. In the past 5 years, 275 lots containing 15,488 RDT kits for malaria diagnosis were subjected to LT. The monovalent RDTs (n = 1,216), based on either Pf histidine rich protein 2 (HRP2) or Pan-Plasmodium lactate dehydrogenase (Pan-pLDH) antigens, showed 90.4% sensitivity and 100% specificity, whereas RDTs based on HRP2 + Pan-pLDH or HRP2 + pLDH (n = 13,924) had sensitivity 95.6% and specificity 99.5%, respectively. RDTs based on PfHRP2 + Pv-pLDH + Pan-pLDH (n = 348) had 100% sensitivity and specificity. In a comparison between HRP2 + pLDH or HRP2 + Pan-pLDH to HRP2 + pLDH + Pan-pLDH RDTs, it was found that the sensitivity of PfHRP2 with Pan-pLDH RDTs (n = 2,382) was only 83%. Of the 275 lots analyzed, 15 lots of PfHRP2 with Pan-pLDH were deferred. The QC panel for Pf revealed a faint Pan band in the tested lots, which is a cause for concern. The results of deferred lots were reported to concerned government agencies. Quality-compromised RDTs may lead to an incorrect diagnosis. It is critical to have a QC system in place for effective malaria management.
Dengue is an acute febrile illness caused by positive-sense single-stranded RNA virus, belonging to the family Flaviviridae and genus Flavivirus. Transmission of virus among the individuals occurred ...by blood-feeding Aedes mosquitoes. This virus has four serotypes differentiated on the basis of antibody neutralization assay. At present, there is no particular treatment or vaccine candidate available for dengue infection. Approximately 3.9 billion human populations are at risk of dengue virus (DENV) infection. Thus, precise diagnosis of dengue at the early stage is very essential for disease control and effective therapy in order to treat or prevent severe complications. Indeed, the accurate diagnosis of DENV remains a problem because of low detection accuracy along with high testing price. Sensitivity and specificity of available kits vary from test to test, and cross-reactivity with other Flavivirus is a challenging issue for diagnosis. In this study, linear epitopes of envelope (E) and NS1 proteins were identified to diagnose the DENV. Whole protein sequences of E and NS1 of DENV were obtained from UniProtKB database. On the basis of algorithm prediction from DNASTAR, BCEPRED, and IEDB data resources, twelve peptides of E (EP1 to EP12) and eight peptides of NS1 (NS1-1 to NS1-8) were selected, which were common in all serotypes. Sequence homologies of peptides with other Flavivirus were checked by Multiple Sequence Alignment Tool ClustalX2. Peptide sequences were synthesized chemically by solid-phase peptide synthesis technique. Dengue-specific IgM and IgG (secondary response) antibodies in the patient’s antisera were tested with the peptides using ELISA protocol. Peptides EP1, EP2, EP4, EP7, EP10, and EP12 of E protein and NS1-1, NS1-3, NS1-4, NS1-7, and NS1-8 of NS1 protein were considered the best immunoreactive peptides with the sensitivity (73.33-96.66%) and specificity (82.14-100%). Such peptides together can be used to construct the multiple antigen peptides (MAP) or multiplexed microbeads for designing a precise, cost-effective, and easy-to-make peptide-based immunodiagnostic kit for DENV detection.
Quality control testing of the vaccine for lot release is of paramount importance in public health. A recent pandemic caused by the SARS-CoV-2 virus brought together all spheres of vaccine to combat ...the virus. The scientific advancement in the development of vaccines facilitated the scientists to develop the vaccine against SARS-CoV-2 in a record time. Thus, these vaccines should be stringently monitored for their safety and efficacy as per the latest WHO and national regulatory guidelines, and quality control evaluation of the product should be done at national control laboratories before releasing the product into the market as it assures the quality and safety of the vaccine.
The SARS-CoV-2 exploited the ACE2 (Angiotensin Converting Enzyme 2) receptor, a surface protein on mammalian cells to gain entry into the host cells. The viral surface protein that interacted with the ACE2 receptor is the Spike protein of SARS-CoV-2. Thus, in the development of the vaccine and assessing its quality, the Spike protein of SARS-CoV-2 became an attractive immunodominant antigen. In National Institute of Biologicals, an apex body in the testing of biologicals in India, received the Adenovector (Adenovirus + vector) based COVID-19 vaccine, a finished product for quality evaluation. Due to the lack of a pharmacopeial monograph, the testing of the vaccine was done as per the manufacturer's specifications and methods. The routine assays of identification employed by the manufacturer do not reflect the expression of Spike protein which is required for the immune system to get activated. In this report, we showed the determination of Spike protein expression by immunoblotting and immunofluorescence for identification parameters in the quality testing of the COVID-19 vaccine. We determined the translation of the SARS-CoV-2 Spike gene cloned into an Adenovector.
The results from these experiments indicated the expression of Spike protein upon infection of mammalian cells with viral particles suggested that the expression of immunodominant Spike protein of SARS-CoV-2 may be employed by quality control laboratories as a parameter for identification.
The study suggested that the determination of the expression of Spike protein is pertinent to identifying the Adenovector based vaccines against COVID-19.
We sequenced and annotated the genomes of four P. vivax strains collected from disparate geographic locations, tripling the number of genome sequences available for this understudied parasite and ...providing the first genome-wide perspective of global variability in this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of P. falciparum, a malaria-causing parasite that results in higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax relative to P. falciparum, which is thought to have undergone a recent population bottleneck. The SNP diversity, as well as additional microsatellite and gene family variability, suggests a capacity for greater functional variation in the global population of P. vivax. These findings warrant a deeper survey of variation in P. vivax to equip disease interventions targeting the distinctive biology of this neglected but major pathogen.
The Comprehensive Case Management Project (CCMP), was a collaborative implementation research initiative to strengthen malaria early detection and complete treatment in Odisha State, India.
A two-arm ...quasi-experimental design was deployed across four districts in Odisha, representing a range of malaria endemicity: Bolangir (low), Dhenkanal (moderate), Angul (high), and Kandhamal (hyper). In each district, a control block received routine malaria control measures, whereas a CCMP block received a range of interventions to intensify surveillance, diagnosis, and case management. Impact was evaluated by difference-in-difference (DID) analysis and interrupted time-series (ITS) analysis of monthly blood examination rate (MBER) and monthly parasite index (MPI) over three phases: phase 1 pre-CCMP (2009-2012) phase 2 CCMP intervention (2013-2015), and phase 3 post-CCMP (2016-2017).
During CCMP implementation, adjusting for control blocks, DID and ITS analysis indicated a 25% increase in MBER and a 96% increase in MPI, followed by a -47% decline in MPI post-CCMP, though MBER was maintained. Level changes in MPI between phases 1 and 2 were most marked in Dhenkanal and Angul with increases of 976% and 287%, respectively, but declines in Bolangir (-57%) and Kandhamal (-22%). Between phase 2 and phase 3, despite the MBER remaining relatively constant, substantial decreases in MPI were observed in Dhenkanal (-78%), and Angul (-59%), with a more modest decline in Bolangir (-13%), and an increase in Kandhamal (14%).
Overall, CCMP improved malaria early detection and treatment through the enhancement of the existing network of malaria services which positively impacted case incidence in three districts. In Kandhamal, which is hyperendemic, the impact was not evident. However, in Dhenkanal and Angul, areas of moderate-to-high malaria endemicity, CCMP interventions precipitated a dramatic increase in case detection and a subsequent decline in malaria incidence, particularly in previously difficult-to-reach communities.
•Robust lyophilized technique for detection of dengue using isothermal process.•Eliminate the need of cold chain for the transportation and storage of chemicals.•Ideal alternative for existing LAMP ...method with a POC approach.•Dengue diagnose within 35 min with least chemicals.•L-LAMP showed better sensitivity and specificity than RT-PCR and ELISA.
Dengue virus infects millions of the people globally each year and its diagnosis remains a challenge. Conventionally used diagnostic methods are complex and time consuming. LAMP technique is a potential alternative for diagnosis of dengue virus. The benefits of LAMP are its ease and ability, as it does not require an expensive equipment and results are effortlessly visualized by the naked eye. However, it does not aid as point of care technique owing to need of contamination free area, deep freezer for chemical storage and primer self amplification. Each small modification in LAMP method bring it towards an ideal point of care technique. An advanced lyophilized loop mediated isothermal amplification (L-LAMP) was developed in which the dye was dried on the cap and reaction reagents was lyophilized at the bottom of the tube to overcome the common hurdles of LAMP technique. The technique was able to diagnose disease within 35 min with 4U of Bst polymerase. The least concentration of dye required was 1000×.
Result given by the seminested reverse transcriptase polymerase chain reaction (RT-PCR) and L-LAMP with enzyme linked immuno sorbent assay (ELISA) were compared using Chi square test. The L-LAMP showed 100 % specificity and 92 % sensitivity with respect ELISA and was found better than RT-PCR which showed 100 % specificity and 88 % sensitivity. There was no cross reactivity of primers with other disease like malaria caused by Plasmodium falciparum and P. vivax and with viral disease chikungunya. L-LAMP has dynamic potential as point of care technique.
Epidemiology of Plasmodium vivax Malaria in India Anvikar, Anupkumar R; Shah, Naman; Dhariwal, Akshay C ...
The American journal of tropical medicine and hygiene,
12/2016, Volume:
95, Issue:
6 Suppl
Journal Article
Peer reviewed
Open access
Historically, malaria in India was predominantly caused by Plasmodium vivax, accounting for 53% of the estimated cases. After the spread of drug-resistant Plasmodium falciparum in the 1990s, the ...prevalence of the two species remained equivalent at the national level for a decade. By 2014, the proportion of P. vivax has decreased to 34% nationally, but with high regional variation. In 2014, P. vivax accounted for around 380,000 malaria cases in India; almost a sixth of all P. vivax cases reported globally. Plasmodium vivax has remained resistant to control measures, particularly in urban areas. Urban malaria is predominantly caused by P. vivax and is subject to outbreaks, often associated with increased mortality, and triggered by bursts of migration and construction. The epidemiology of P. vivax varies substantially within India, including multiple relapse phenotypes with varying latencies between primary infection and relapse. Moreover, the hypnozoite reservoir maintains transmission potential and enables reestablishment of the parasite in areas in which it was thought eradicated. The burden of malaria in India is complex because of the highly variable malaria eco-epidemiological profiles, transmission factors, and the presence of multiple Plasmodium species and Anopheles vectors. This review of P. vivax malaria in India describes epidemiological trends with particular attention to four states: Gujarat, Karnataka, Haryana, and Odisha.
•A magnetic multiplex LAMP technique is developed to diagnose two diseases with similar symptoms.•Advance of LAMP method with a POC approach.•Dengue and chikungunya diagnose within 45 ...min.•Sensitivity of MM LAMP visualization was 51.65 ng/μl amplified DNA.
Dengue and chikungunya viruses are arthropod borne virus spread through common vector instigating infection in human. There has been an increased recognition that more attention needs to be paid to similar sympotoms caused by both of the virus as they spread in the same region at same time. It warrants need of cost effective, user friendly and rapid multiplex diagnostic technique which could simultaneously diagnose and identify between two virus diseases in resource poor setting. A magnetic multiplex loop mediated isothermal amplification (MM-LAMP) technique was developed by coupling multiplex LAMP with magnetic particle-based naked eye visualization to overcome the shortcoming of simultaneous detection of both diseases. In recent years this technology has emerged as a particularly attractive candidate as amplification reaction process completes within 45 min. The first step involves multiplexing biotin and digoxigenin coated dengue and chikungunya primers respectively in LAMP reaction followed by precipitation of the amplified DNA with polyethylene glycol (PEG) buffer and finally clumping with streptavidin and anti-digoxigenin coated magnetic particle for virus discrimination and naked eye visualization. The DNA detection limit of MM LAMP visualization was 51.65 ng/μl which is comparable to the electrophoresis base UV light visualization. The results showed potential superiority over standard methods polymerase chain reaction (PCR). This current advancement empowers multiplex LAMP utility in resource limited setting without using any of the florescent dyes, turbidimeter, or the sophisticated quantitative PCR machine etc which restrict multiplex LAMP technique to laboratorial use only. We have proposed a novel method without such limitations. This technique has potential as a point of care technique for simultaneous detection of two diseases.
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
species. Approximately 30% of randomly selected samples (
= 586) were analyzed using real-time PCR (qPCR), and the genetic diversity of
was analyzed. The prevalence of
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
,
, and mixed infection (
and
) 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 (
) and (
), respectively. Expected heterozygosity was 0.64 and 0.47 for
and
, 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.
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