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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusions
 ~  References
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BRIEF COMMUNICATION
Year : 2017  |  Volume : 35  |  Issue : 3  |  Page : 410-414
 

Epidemiology of malaria and MSP-2 gene-based genetic diversity of Plasmodium falciparum from patients attending community health centre, Jiribam, Manipur


Department of Microbiology, Assam University, Silchar, Assam, India

Date of Web Publication12-Oct-2017

Correspondence Address:
Indu Sharma
Department of Microbiology, Assam University, Silchar - 788 011, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_14_532

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 ~ Abstract 

Two millilitres of peripheral blood was collected from 323 patients of different age groups and rapid diagnosis (RDT test) was performed. Parasite genomic DNA was extracted from whole blood and MSP-2 gene-based diversity and polymorphism was determined followed by restriction fragment length polymorphism analysis using Taq 1 and Vsp 1 digestion of each polymerase chain reaction product to analyse MSP-2 genotypes. Twenty-six sequences of P. falciparum MSP-2 gene were retrieved from the current GenBank database to represent strains from various geographical locations and were analysed for the Taq 1 and Vsp 1 enzyme restriction sites using in silico restriction digestion in Sequence Manipulation Suite, Version 2.


Keywords: Community health centre, MSP-2 genotypes, P. falciparum, restriction fragment length polymorphism


How to cite this article:
Sharma I, Saikia A, Chakraborty P. Epidemiology of malaria and MSP-2 gene-based genetic diversity of Plasmodium falciparum from patients attending community health centre, Jiribam, Manipur. Indian J Med Microbiol 2017;35:410-4

How to cite this URL:
Sharma I, Saikia A, Chakraborty P. Epidemiology of malaria and MSP-2 gene-based genetic diversity of Plasmodium falciparum from patients attending community health centre, Jiribam, Manipur. Indian J Med Microbiol [serial online] 2017 [cited 2018 Oct 22];35:410-4. Available from: http://www.ijmm.org/text.asp?2017/35/3/410/216610



 ~ Introduction Top


India has the largest population in the world at risk of malaria, with 85% of total population living in malarious zones. In most endemic areas, Plasmodium vivax cohabits with Plasmodium falciparum, so mixed infections with these two species are common. P. vivax is more difficult to control and eliminate than P. falciparum because of its tendency to relapse after resolution of the primary infection. In endemic areas, relapse of P.vivax-associated malaria is a major cause of malaria in young children and an important source of malaria transmission.[1]

In India, malaria is endemic in most parts where insecticide resistance among malaria vectors, drug resistance in malaria parasites and lack of adequate resources pose serious challenges for fighting malaria.[2] Nine species of Anopheles mosquitoes are responsible for malarial transmission in India, of which six are of primary importance.[3]

P. falciparum exhibits extensive genetic diversity and is likely to reduce the effectiveness of the subunit vaccines and emergence of drug resistant that are used against the key parasite. Sequence polymorphisms have also been described for MSP-2 genes by various workers;[4] however, little is known about the extent of the genetic repertoire of naturally circulating strains of Plasmodium in different geographical areas of northeastern states where malaria is endemic. Moreover, the northeastern states have reported maximum cases of malaria, and of these, Manipur is one of the leading states in malarial field outbreaks.[5] However, no information about frequency of field outbreaks in the studied population by molecular methods have encountered in Manipur parasite population yet. The present study was, therefore, designed to ascertain the occurrence of malaria cases in human and to determine the genetic diversity of the field isolates tested positive for Plasmodium species by immunochromatographic assay, uniplex polymerase chain reaction and restriction fragment length polymorphism (RFLP) assays. This study will thus highlight the prevalence of malaria incidence cases and genetic perplexity of Plasmodium circulating among the human population that would have implications in surveillance and vaccine evaluation program.


 ~ Materials and Methods Top


Two millilitres of peripheral blood was collected from 323 patients of different age groups, suspected to be suffering from malaria and immediately transferred to the EDTA-coated specimen vial.

Rapid diagnosis (RDT test) was done using BIOLINE SD Rapid test Kit (Standard Diagnostics Inc., Gurgaon, Haryana, India) as per manufacturer's instruction. In the present study, the epidemiology and prevalence of different Plasmodium species were also studied. Data based on sex, age and season were obtained and analysed.

Parasitic genomic DNA extraction and polymerase chain reaction amplification of MSP-2 gene

Parasite genomic DNA was extracted from whole blood as described by Sambrook et al., 1989.[6] The polymerase chain reaction (PCR) procedure consisted of preparation of master mix containing 10 × Taq buffer (Thermo Scientific, USA), 10 mM dNTPs mix, 25 mM MgCl2, 20 pMol of each primer and 0.5U of Rec Taq DNA polymerase (Thermo Scientific, USA) in a final volume of 20 μl. The reaction mixtures were denatured at 95°C for 5 min, followed by 35 amplification cycles at 95°C for 30 s, 58°C for 1 min, 68°C for 2 min and a final extension at 68°C for 5 min. The PCR-amplified products were analysed in 1% agarose gels containing ethidium bromide (0.5 mg/ml) in 0.5 × tris-borate-EDTA buffer. The amplified products were stored at −40°C. Simplex primary PCR (Master Cycler Gradient, Eppendorf) was carried out using primers MSA 2 for the conserved region. The primer sequences used were MSA 2-F-5′-GAA GGT AAT TAA AAC ATT GTC-3′ and MSA 2-R-5′-GAG GGA TGT TGC TGC TCC ACA G- 3′.[7]

Polymerase chain reaction-restriction fragment length polymorphism analysis of MSP-2 Genotypes

RFLP analysis was achieved using Taq 1 and Vsp 1 digest of each PCR product to analyse MSP-2 genotypes. In Taq1 and Vsp1 restriction digestion, 10 μl of PCR product was digested with the respective restriction enzyme in a total reaction volume of 15 μl. Further, a 10 μl of PCR product was combined with 3 μl of nuclease free water, 10 × buffer and 0.5 U of respective enzymes. The contents were mixed adequately by short spin and then incubated at 37°C for 2 h.

Agarose gel electrophoresis and product detection

Total volume of each RFLP product was loaded on a 2% agarose gel containing ethidium bromide and electrophoresed in 1 × tris-acetate-EDTA buffer at an appropriate electric field for optimum separation. DNA bands were visualised under ultraviolet light using gel documentation system (DNR Bio-imaging System). Genetic diversity was identified by fragment length position and the sizes of the PCR products were estimated using a 100 base pair ladder.

Data analysis

Data analysis was based on the visual inspection of amplified fragments separated by 2% agarose gel electrophoresis. MSP-2 gene-based diversity and polymorphism were determined based on the number of fragments and the size of each fragment. Fragment sizes were estimated by comparing with molecular size standards of a 100 base pair DNA ladder.

Comparison with global isolates

Twenty-six sequences of P. falciparum MSP-2 gene were retrieved from the current GenBank database to represent strains from various geographical locations. The sequences were analysed for the

Taq 1 and Vsp 1 enzyme restriction sites using in silico restriction digestion in Sequence Manipulation Suite, Version 2. (www.bioinformatics.org/sms2/) The in silico restriction digestion results were then compared with the restriction pattern of local isolates.


 ~ Results Top


Prevalence of different Plasmodium species among screened samples

P. falciparum parasites were detected in a total of 159 patients out of 323 samples showing an overall prevalence of 49.2%. Of the 159 positive samples, 84 samples (52.83%) were found to be positive for P. falciparum; 47 samples (29.56%) were positive for P. vivax and 28 cases (17.61%) were found to be positive for P. falciparum/P. vivax coinfection indicating that the prevalence of P. falciparum is significantly higher followed by P. vivax and P. falciparum/P. vivax coinfection.

Relationship between sex and prevalence of Plasmodium falciparum, Plasmodium vivax and Plasmodium falciparum, Plasmodium vivax coinfection

The relationship between sex and prevalence of different plasmodium infection was analysed and it was found that of the total 84 P. falciprum positive samples, 47 (55.6%) males and 37 (44.4%) females were infected with the malarial parasite and of 47 P. vivax positives, 25 (53.2%) were males and 22 (46.8%) females. Furthermore, a P. falciprum/P. vivax coinfection was also observed in 28 positive samples, of which 17 (60.7%) were male hosts and 11 (39.3%) were female hosts

Occurrence and prevalence of Plasmodium infection (Plasmodium falciparum, Plasmodium vivax, Plasmodium falciparum + Plasmodium vivax) with respect to age group and seasonal changes

The prevalence of different Plasmodium infection in different age groups was determined. It was found that the age group of 31–40 was most prevalent and vulnerable (63%), followed by the age group of 21–30 (61%) and season-wise variation of Plasmodium infection revealed that cases of malaria increased with the commencement of rainy season when compared to spring, autumn and winter seasons.

Polymerase chain reaction amplification of MSP-2 gene of Plasmodium falciparum

The field samples positive for P. falciparum were selected for genomic DNA isolation. Of the 84 samples, only 60 samples (71%) revealed P. falciparum DNA and 35 samples were subjected to primary PCR for amplification of MSP-2 gene. The product size ranged of 460 base pairs with a single band [Figure 1]. A total of 14 PCR products were generated out of 35 field samples and the quantitative estimation gave result for each product in the range of 80–150 ng/μl. The overall prevalence using MSP-2 gene-based PCR was estimated of 40%.
Figure 1: Amplification products corresponding to the Msp-2 gene of Plasmodium falciparum from human blood samples are shown. Lane M = molecular weight markers (100-bp ladder); C = Control; Lane 1 = Sample MP-1; Lane 2 = Sample MP-2; Lane 3 = Sample MP-3; Lane 4 = Sample MP-4, Lane 5 = Sample MP-5; Lane 6 = Sample MP-6; Lane 7 = Sample MP-7; Lane 8 = Sample MP-8; Lane 9 = Sample MP-9. The size of amplified bands is indicated

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In vitro restriction fragment length polymorphism assay analysis of amplified MSP-2 gene products

The amplified PCR products were subjected to restriction digestion using Taq I (TlCGA) and Vsp I (AT∣TAAT) restriction enzymes. MSP-2 gene-based diversity and polymorphism were determined based on the number of fragments and the size of each fragment. Restriction enzyme digested MSP-2 gene products generated from P. falciparum field isolates were analysed by agarose gel (2%) electrophoresis. Both the enzymes in each case produced consistent restriction profiles. The corresponding banding pattern or enzyme profiles were defined for each restriction enzyme with profiles T and V for enzymes TaqI and VspI, respectively. The fragment arrays thus generated were correlated with band patterns generated from the current database by similar enzymes as described to determine the characteristic similarity of Indian global isolates.

A total of five restriction profiles were obtained by digestion of msp-2 gene from 14 P. falciparum strains with Vsp Ienzyme[profiles V1–V5; [Figure 2]. Further, 64% of the strains could not demonstrated Vsp I cleavage site and designated to enzyme profile V1, whereas empirical analysis showed characteristic banding patterns in the particular gene of conserved nucleotide stretches. Isolates MP2 and MP3 have demonstrated similar enzyme profile and were designated to single enzyme profile V2. Profile V3, V4 and V5 were found associated with single isolates and having unique restriction band patterns.
Figure 2: Amplified Msp-2 gene segment enzyme profiles of representative Plasmodium falciparum field isolates out of the total collection was shown here. The entire isolates demonstrated consistent patterns after digestion with restriction enzyme, Taq I

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Similarly, five restriction profiles were obtained after digestion of msp-2 gene products with Taq I and designated as T profiles [profiles T1 to T5; [Figure 3]. Each strain revealed consistent and in some cases, additional cleavage site representing variation in the enzyme restriction sites. Few isolates (50%) have demonstrated no cleavage patterns for Taq I restriction enzyme and designated to enzyme profile T5 for all the associated strains. The enzyme profile T1 was found associated with 21.4% of isolates; however, enzyme profile T2 and T3 was found unique demonstrated by two single strains of different epidemics. About 14.2% of P. falciparum strains were associated with single banding pattern T4.
Figure 3: Amplified Msp-2 gene segment enzyme profiles of representative Plasmodium falciparum field isolates out of the total collection was shown here. The entire isolates demonstrated consistent patterns after digestion with restriction enzyme, Vsp I

Click here to view


In silico restriction fragment length polymorphism assay analysis

An in silico RFLP assay was designed for twenty-six global isolates using the same restriction enzymes as inin vitro analysis to demonstrate the genetic variations among the global isolates. Maximum isolates showed no cleavage patterns for Vsp I and thus were designated as V1 profile. An additional V profile V6 by GBS-26 strain was observed after Vsp I digestion. This profile was distinct from the associated local strains Vsp I enzyme profile. Similarly, a large set of isolates (92%) found to be associated with profile T5 exhibited no restriction sites within the targeted gene. However, one distinct T profile (T6) was related with the database strain (GBS-6) which was obtained after computational digestion of the retrieved global strains by Taq Irestriction enzyme which was found related with profile T2 when compared with the local strains.

Combined restriction fragment length polymorphism profile

The generated enzyme profiles of both local and global isolates were combined together to produce characteristic RFLP pattern for each strain. The 40 P. falciparum Msp-2 encoding genes were classified into a total of 10 RFLP patterns, of which only one pattern was observed among the local strains. Majority of the RFLP combination was found specifically unique among both the local and global isolates. None of the isolates shared similar patterns within themselves and the RFLP profiles of the global strains showed marked variations through local isolates. About 42% of local isolates exhibited a unique profile while the others (57%) were associated with single pattern. However, the global strains, GBS-5 and GBS- 26 isolates represented a unique pattern of RFLP (V6T2) from the other isolates.


 ~ Discussion Top


Molecular analysis of parasites collected in this survey demonstrates that parasite multiplicity is very common in this community along with the multiple P. falciparum genotype infections.[13] P. falciparum, a major malaria parasite, found in this region is responsible for almost 60-80% of infections along with the hot and humid climate prevailing in this region[5] making more idealistic for its survival and multiplication.[8],[9]

The different Plasmodium species among screened samples revealed 52.83% prevalence of P. falciparum infection, of which 29.56% were positive for P. vivax and 17.61% for P. falciparum/P. vivax coinfection thus suggesting that the prevalence of P. falciparum was significantly higher in the study site followed by P. vivax and P. falciparum/P. vivax coinfection.[10] Although very restricted reports on the prevalence of Plasmodium species with respect to sex, age and season are available, in the present study, it was found that the age group of 31–40 was the most prevalent and vulnerable (63%), followed by the age group of 21–30 (61%) to infections. The burden was observed to be generally higher in men than in women in all age groups.[11] About 56% of male and 44% female patients were found positive for overall Plasmodium infection in Manipur. No significant difference between male and female regarding the infection of P. vivax and P. falciparum/P. vivax coinfection was observed; while seasonal variation interfered with the severity of cases of malarial infections with the commencement of rainy season when compared to spring, autumn and winter seasons.[5] Highest numbers of infection were recorded in the months of April–June.

The present study is the first to provide information about the genetic diversity of P. falciparum in Manipur. A total of 84 P. falciparum positive samples were subjected to DNA isolation and only 60 samples (71%) revealed P. falciparum DNA. Further, samples subjected to primary PCR for amplification of MSP-2 gene demonstrated overall prevalence of 40% positive cases.

The present study of MSP-2 gene-based variation of P. falciparum; the RFLP assay demonstrated high MSP-2 gene diversity and polymorphism.[4] The polymorphism within the important molecular marker of P. falciparum, i.e. Msp-2 gene, has shown to be an important mechanism to generate diversity on many occasions, and moreover, other less important mechanisms such as inter- or intra-genic recombination are believed to occur less frequently. Hence, the affinity of Plasmodium field strains toward high genetic diversity could significantly boost the evolution and subsequent emergence of novel strains.[12]

Comparison of RFLP data for local isolates to those of global origin suggested the local Plasmodium population is heterozygous and distinguishable from global strains. All the local and global strains produced a consistent profile through empirical analysis, and moreover, except few, maximum isolates during the study period exhibited a greater diversity by providing additional or no cleavage site. Thus, it may be assumed that there is point mutation occurring in the gene during replication and the existing restriction enzyme sites have been or new sites have generated. Hence, considerable genetic variation between global and local isolates was established and none of their associated RFLP patterns were identified in the local strains excluding one enzyme patterns of uncut restriction profile constructing similarity with that of global origin.


 ~ Conclusions Top


The focus of the present study was to determine the epidemiology of malaria in different group of people attending the Community Health Centre, Jiribam, and to study the genetic diversity of P. falciparum by PCR-RFLP method. The study site was observed to be coendemic region for P. falciparum and P. vivax- associated malaria. Perennial and persistent transmission of the disease with increased infection during March–June with the occurrence of rain was observed. The field isolates were highly diverse in respect of MSP-2 gene with identical population structure and exhibited a level of diversity with low to mesoendemicity of malaria thus, reinforcing the value of these genotyping markers in classifying recurrent post-treatment P. falciparum episodes as recrudescence or new infections. However, the study is lacking the sequence information of Msp-2 which is in generation. With the current changes in malaria epidemiology, the P. falciparum msp1and glurp allele frequency/genetic diversity should be monitored regularly along with msp-2 gene to ensure the reliability of the PCR-adjusted treatment outcome.

Acknowledgments

We wish to acknowledge the cooperation of the Community Health Centre, Jiribam (Manipur).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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Mobegi VA, Loua KM, Ahouidi AD, Satoguina J, Nwakanma DC, Amambua-Ngwa A, et al. Population genetic structure of Plasmodium falciparum across a region of diverse endemicity in West Africa. Malar J 2012;11:223.  Back to cited text no. 12
    
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Mwingira F, Nkwengulila G, Schoepflin S, Sumari D, Beck HP, Snounou G, Felger I, Olliaro P, Mugittu K. Plasmodium falciparum msp1, msp2 and glurp allele frequency and diversity in sub-Saharan Africa. Malar J 2011; 10: 79.  Back to cited text no. 13
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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