Genetic diversity and multiplicity of infection of Plasmodium falciparum isolates from Kolkata, West Bengal, India Pabitra Saha a,b, ⁎, Swagata Ganguly a,c , Ardhendu K. Maji a a Calcutta School of Tropical Medicine, 108, C. R. Avenue, Kolkata 700 073, India b Department of Zoology, A. P. C. Roy Govt. College, Himachal Bihar, Matigara, Siliguri, 734010, West Bengal, India c Department of Microbiology, N. R. S. Medical College & Hospital, Kolkata, West Bengal, India abstract article info Article history: Received 11 April 2016 Received in revised form 28 May 2016 Accepted 30 May 2016 Available online 31 May 2016 The study of genetic diversity of Plasmodium falciparum is necessary to understand the distribution and dynamics of parasite populations. The genetic diversity of P. falciparum merozoite surface protein-1 and 2 has been exten- sively studied from different parts of world. However, limited data are available from India. This study was aimed to determine the genetic diversity and multiplicity of infection (MOI) of P. falciparum population in Kolkata, West Bengal, India. A total of 80 day-zero blood samples from Kolkata were collected during a therapeutic efficacy study in 2008– 2009. DNA was extracted; allelic frequency and diversity were investigated by PCR-genotyping method for msp1 and msp2 gene and fragment sizing was done by Bio-Rad Gel-Doc system using Image Lab (version 4.1) software. P. falciparum msp1 and msp2 markers were highly polymorphic with low allele frequencies. In Kolkata, 27 msp1 different genotypes (including 11of K1, 6 of MAD20 and 10 of Ro33 allelic families) and 30 different msp2 geno- types (of which 17 and 13 belonged to the FC27 and 3D7 allelic families, respectively) were recorded. The major- ity of these genotypes occurred at a frequency below 10%. The mean MOI for msp1 and msp2 gene were 2.05 and 3.72, respectively. The P. falciparum population of Kolkata was genetically diverse. As the frequencies of most of the msp1 and msp2 alleles were low, the probability of new infection with genotype identical to that in pretreatment infection was very rare. This information will serve as baseline data for evaluation of malaria control interventions as well as for monitoring the parasite population structure. © 2016 Elsevier B.V. All rights reserved. Keyword: Genetic diversity Multiplicity of infection (MOI) Plasmodium falciparum msp1 msp2 1. Introduction Malaria remains the most important public health disease in the tropical and subtropical parts of the world and more than 40% of the world's population is still at risk of infection (Hay et al., 2004). World Health Organization (WHO) reported about 214 million malaria cases world-wide as well as 438,000 deaths in 2014, especially in sub-Saharan Africa (WHO, 2015). Plasmodium falciparum is the most virulent species that infects human and is responsible for most malaria related deaths. In recent years several control measures have been introduced to reduce malaria-related morbidity and mortality which mainly comprise of in- door residual spray (IRS), insecticide treated bed nets (ITNs) and che- motherapy. But a universal effective malaria vaccine is considered to be needed in addition to the existing malaria control strategies for better controlling and eventually eradicating malaria from the globe (Kilama and Ntoumi, 2009; Thera and Plowe, 2012; Hill, 2011; Schwartz et al., 2012). The major obstacles in the development of an ef- fective malaria vaccine are antigenic variation presented by P. falciparum due to genetic diversity, occurrence of variant forms of the parasite in different geographical areas, and multiplicity of infection (MOI) i.e., multiple genotypes in a single infection (Thera and Plowe, 2012; Raj et al., 2004; Mahajan et al., 2005). The study of genetic diver- sity is important to understand the distribution, dynamics and genetic structure of prevailing parasite population. Two polymorphic genes - merozoite surface protein-1 (msp1) and merozoite surface protein-2 (msp2) of P. falciparum were extensively studied by different workers for this purpose (Ranjit and Sharma, 1999; Haddad et al., 1999; Raj et al., 2004; Mahajan et al., 2005; Zakeri et al., 2005; Silue et al., 2006; Soulama et al., 2009; Ghanchi et al., 2010; Kang et al., 2010; Mwingira et al., 2011; Atroosh et al., 2011; Mohammed et al., 2015; Yavo et al., 2016). MSP1 is a major surface protein of approximately 190-kDa size and plays a major role in erythrocyte invasion (Holder et al., 1992). The Infection, Genetics and Evolution 43 (2016) 239–244 ⁎ Corresponding author at: Department of Zoology, A. P. C. Roy Govt. College, Himachal Bihar, Matigara, Siliguri, 734010, West Bengal, India. E-mail addresses: pabitra.saha82@gmail.com (P. Saha), swagatamedicine@gmail.com (S. Ganguly), maji_ardhendu@yahoo.com (A.K. Maji). http://dx.doi.org/10.1016/j.meegid.2016.05.038 1567-1348/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid