Genetic polymorphism in Plasmodium falciparum: Differentiation of parasite isolates of high & low virulence by RAPD.

Background & objectives: The increase in Plasmodium falciparum infections which are associated with severe and complicated malaria and drug resistance has made control of malaria a difficult task. Extensive genetic polymorphism in P. falciparum has been reported from several parts of the world which affects the efficacy of sub-unit vaccines. The knowledge of genotypes of the parasite in a geographical region is therefore, important for effective management and control. The aim of the present study was to investigate the usefulness of random amplified polymorphic DNA (RAPD)-PCR technique for differentiation of P. falciparum isolates from patients presenting with severe (cerebral malaria) and mild malaria. Methods: Genetic polymorphism in 21 P. falciparum isolates obtained from patients found positive for P. falciparum by light microscopy was studied by RAPD-PCR analysis. Eleven RAPD primers were used for analysis of 21 P. falciparum isolates obtained from cerebral and non-cerebral malaria patients. Results: Of the 11 primers, only three (E-4, E-8, and R-8) produced useful polymorphic patterns. The cluster analysis based on UPGMA demonstrated that isolates causing cerebral malaria cluster separately from those causing uncomplicated malaria. However, the analysis of phylogenic tree showed that P. falciparum isolates causing non-cerebral and cerebral malaria clustered separately but showed relatedness. Interpretation & conclusions: The results of the present study showed that the RAPD-PCR was able to differentiate the isolates causing severe and mild malaria. The cluster analysis of the phylogenic tree suggested that the virulent strains evolved from less virulent strains as it clustered separately. RAPD technique may be useful in discriminating between the different isolates of the same species resulting in different clinical profiles.

Polymorphism in merozoite surface protein-1 gene in north & northwest indian field isolates of Plasmodium vivax.

Background & objective: Merozoite surface protein-1 of Plasmodium vivax (Pvmsp-1) is a strong vaccine candidate against asexual blood stages. Extensive polymorphism in msp-1 gene has been reported in P. vivax isolates from different geographical regions which is necessary before a field trial of any malaria vaccine based on msp-1 is undertaken. There are only a few reports available on polymorphism in msp-1 gene in Indian field isolates of P. vivax. The aim of the present study was therefore to investigate the polymorphism in Pvmsp-1 gene in 25 isolates of P. vivax collected from malaria patients from regions of north and northwest India. Methods: Parasite DNA was extracted from whole blood samples collected in citrated anticoagulant. The polymorphic region-5, the most variable region of the Pvmsp-1 gene was amplified by PCR. The PCR products were further analyzed by restriction fragment length polymorphism (RFLP) using Mva-1 restriction enzyme. The DNA fragments obtained on PCR and RFLP were analyzed by agarose gel electrophoresis. Results: On the basis of PCR, significant size polymorphism was seen and 4 allelic types were observed amongst the 25 isolates. Further analysis by RFLP discriminated these 4 allelic types into 9 sub-allelic types indicating that PCR-RFLP can be a good tool to study polymorphism in msp-1 gene of Plasmodium. Interpretation & conclusion: Marked genetic polymorphism was observed in msp-1 gene among the isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax.

Genetic Polymorphism in msp-2, ama-1 and csp genes in Plasmodium falciparum field isolates from north and north-western India.

Background & objectives: Malaria is a major public health problem in tropical and sub-tropical countries. Malaria vaccine is highly desirable as an adjunct to existing malaria control measures. The polymorphism in vaccine candidate antigens might be a hurdle in developing an effective vaccine. Merozoite surface protein-2, apical membrane antigen-1 and circumsporozoite protein of Plasmodium falciparum are vaccine candidate antigens. The aim of this study was to detect extent of genetic polymorphism in potential vaccine candidate antigen genes, i.e. msp-2, ama-1 and csp of P. falciparum isolates prevalent in northern and north-western parts of India. Methods: Overall 88 parasite isolates of P. falciparum were collected during July 1998–March 2002 from different parts of northern and north-western India. DNA was extracted and analyzed for genetic polymorphism by PCR-RFLP method. For msp-2 gene, family-specific (FC-27 and 3D7) nested PCR was also performed. Results: PCR showed size polymorphism in all the target genes. Three alleles were observed in msp-2 and ama-1, while only two in csp. RFLP of ama-1 and csp with Dra-1 and Ssp-1 endonucleases respectively, failed to differentiate isolates in sub-allelic types, while Hinf-I digestion of msp-2 amplicons differentiated three alleles into two distinct allelic families, i.e. FC-27 and 3D7. The allelic family-specific PCR generally confirmed the results of PCR-RFLP except in a few isolates, which showed mixed (two) clones of msp-2 gene. Interpretation & conclusion: There was extensive polymorphism in msp-2 gene while ama-1 and csp genes showed low polymorphism which may be due to the functional constraints of these proteins. The low level transmission of malaria in the study area may also be a factor for low polymorphism.

Concomitant intestinal parasitism and noncholera vibrio infection.

Concomitant parasitism is not uncommon especially in tropical countries with low socioeconomic status. Here we report an unusual combination of intestinal infection due to Strongyloides stercoralis, Blastomyces hominis and non-cholera Vibrio in a patient suffering from acute gastroenteritis and hypoalbuminemia. Early recognition and accurate treatment of gastrointestinal infections and infestations before the patient develops complications is important.

Genetic polymorphism in Plasmodium falciparum vaccine candidate antigens.

Malaria is still a major public health problem in many tropical and subtropical countries. Malaria vaccine is highly desirable as an adjunct to existing malaria control measures. The polymorphisms in malaria vaccine candidates antigens might be a hurdle in developing an effective vaccine. The present article reviews the genetic polymorphism in several antigens expressed on the parasite surface, which are targets for immunological responses of the host and are good candidates for vaccine development against P. falciparum. Variable regions of most genes are generally dimorphic probably as a result of intragenic recombinations. Each allele in turn shows polymorphism resulting from point mutations, or other mechanisms. Several antigens like merozoite surface protein-1 and 2 (MSP-1 and MSP-2) and S antigen show high polymorphism while in others like circumsporozoite protein (CSP), apical membrane antigen-1 (AMA-1) and erythrocyte binding antigen-175 (EBA-175) functional constraints limit the degree of polymorphism. Polymorphism reported in these genes is discussed.

Association of parasitic infections and cancers.

Recent advances in the fields of molecular biology, epidemiology and infectious diseases have led to significant revelations to clarify the relationship between cancer and infective agents. This article reviews the relationship between parasitic infections and carcinogenesis and the possible mechanisms involved. Few parasites, e.g., Schistosoma haematobium and Opisthorchis viverrini have been found to be strongly associated with bladder cancer and cholangiocarcinoma respectively. The evidence for the association of several other parasites and cancers has also been postulated.

Seroepidemiological study of toxoplasmosis in different sections of population of Union Territory of Chandigarh.

Infections with Toxoplasma gondii in humans are usually asymptomatic or in the form of mild febrile illness. Primary infection in pregnant women may result in congenital toxoplasmosis while infection in immunocompromised subjects like AIDS patients may cause potentially fatal toxoplasma encephalitis. In India, only a few studies in hospital based patients have shown prevalence of toxoplasmosis to be between 1.5 and 21%. No field study involving general population is available. The present study investigates the prevalence of toxoplasmosis in subjects from rural, urban and urban slum populations of Union Territory, Chandigarh. Serum samples from 500 subjects from each group were collected and antitoxoplasma IgM and IgG was detected by conventional micro ELISA technique using soluble Toxoplasma gondii tachyzoite antigen. Overall 5.4% subjects were positive for IgM while 4.66% showed IgG antitoxoplasma antibodies. Amongst the three groups, significantly higher number of subjects in slum area (7.8%) showed IgM antibodies as compared to urban and rural areas (4.2% each). There was no significant difference in IgG positivity between three study areas. Prevalence of T. gondii specific IgG antibodies was significantly higher amongst females of both slum (7.31%) and rural area (8.44%) as compared to the males (2.85% and 3.27% respectively) in the same areas (p<0.05) and also to females of the urban area (2.98%, p<0.05). Prevalence of IgM antibodies was significantly higher (p<0.05) in females in the slum area (10.5%) as compared to females in the urban area (2.55%). In both urban and slum areas, highest IgM seropositivity was observed in age group 6-12 years (10% and 13.3% respectively), while in the rural area the highest IgM seropositivity was seen in the age group > or = 5 years (17.7%). These data indicate that majority of children are exposed to toxoplasma before 12 years of age and particularly in rural areas higher number of subjects acquire Toxoplasma gondii infection early in childhood probably as a result of higher exposure due to farming, poor hygiene and handling of animals.

Comparative evaluation of methods of malaria parasite density determination in blood samples from patients & experimental animals.

Three methods for the quantitation of parasitaemia in malaria were compared with the standard method for ascertaining the accuracy in patients, Plasmodium berghei infected mice and P. knowlesi infected Rhesus monkeys. Technique I, where parasitaemia was calculated from the number of PRBCs in 10,000 RBCs in thin blood film and the total RBC count of the host, was used as the standard. Technique II, where parasitaemia was calculated based on the number of PRBCs per WBC and average total WBC count (8000/microliter), was least accurate. Technique IV, where parasitaemia was calculated from the number of PRBCs per oil immersion field (OIF) of microscope and the estimated amount of blood in one OIF of a thick smear, was most accurate when parasitaemia was low as in malaria patients and experimental animals with < 1 per cent parasitaemia. In mice with moderate parasitaemia (5-10%) and in falciparum malaria cases (with 3-7% parasitaemia) also technique IV was most accurate. In both animal models showing high (15-25%) and in monkeys with moderate parasitaemia, technique III based on the number of PRBCs per WBC and actual total WBC count, was the most accurate. Thus, technique IV being simpler and cost effective, with standardization of the amount of blood used in making a thick smear, may be used routinely for quantitation of parasitaemia.

Biochemical changes induced by malarial parasites.

The morbidity associated with malaria plays a key role in the staggering of the social and economic development of human race. The investigations on the cellular, biochemical and molecular organisation of the malarial parasite are important to understand the host parasite interactions in a better way. The parasite induces several biochemical and biophysical alterations in the host red cells. It is well recognized that cation homeostasis is vital to basic aspects of cell functions. Though the pathogenesis of anaemia associated with Plasmodium falciparum infection is multifactorial, the complex mechanisms involving the role of oxidant stress and calcium imbalance of infected red cells plays an important role.

Increased glutathione cycling and vitamin E of P. falciparum infected erythrocytes fail to prevent spontaneous haemolysis.

In an attempt to understand the pathogenesis of anaemia in Plasmodium falciparum infection, the status of erythrocyte glutathione and vitamin E content in relation to the susceptibility of infected red cells to peroxide haemolysis was examined. Synchronized cultures of the parasite with either ring-, trophozoite or schizont-infected red cells showed a gradual increase in the reduced glutathione content which was significantly higher (p < 0.05) in schizont-infected cells. Trophozoite-infected cells revealed significant increase in oxidized glutathione (p < 0.01) suggesting an increase in glutathione utilization during active erythrocytic schizogony of the parasites. The membrane antioxidant vitamin E also showed an increased accumulation in trophozoite- and schizont-infected red cells (p < 0.05) but not in the uninfected or ring-infected erythrocytes. Despite a favourable change in these antioxidants, the infected as well as uninfected red cells from parasite cultures showed enhanced peroxide haemolysis (uninfected, p < 0.05; ring-rich, p < 0.05, trophozoite- and schizont-rich, p < 0.001). The study provided direct evidence for enhanced susceptibility of red cells to lysis, including those of uninfected cells exposed to parasite products. This might explain the cause for much higher red cell loss and anaemia during P. falciparum infection than all the infected cells put together.

Plasmodial antigen detection by monoclonal antibody as a screening procedure for blood donors in transfusion medicine.

Very little information is available as regards the methods to be advocated to prevent transfusion malaria, especially in endemic countries. Most of the malaria non-endemic countries follow the rule of donor deferral for 3 years after malaria infection. This criterion cannot be followed in endemic areas since the majority of the population is continuously exposed to this infection. Therefore, there was a long felt need for a suitable screening procedure for blood donors to make the transfusion therapy safe. A total of 6,435 blood donors and 3,621 patients who received blood from these donors were studied by blood smears examination and malarial antigen detection by specific monoclonal antibody. Smear examination by Giemsa and acridine orange staining methods showed poor results (0.06% and 0.1% positivity respectively), probably due to low concentration of parasites. However, antigen detection by monoclonal antibody confirmed specific diagnosis in majority of these subjects. Blood smear examination failed to reveal malaria infection in 92.3% of antigen positive blood donors. It is, therefore, recommended that antigen detection by monoclonal antibody should be adopted as a routine screening procedure by the blood transfusion services in malaria endemic countries like India.

Isolation rates of different mycobacterial species from Chandigarh (north India).

A total of 4958 patients, clinically suspected to have tuberculosis were screened for mycobacteria by acid fast staining and culture procedures. Mycobacterial species were isolated from 462 (9.3%) patients while acid fast bacilli were demonstrated on smear examination in 83 (1.7%) patients. Mycobacterium tuberculosis was the most common isolate (92%). Among the nontuberculous mycobacteria, M. fortuitum was isolated in 13 (2.8%), M. avium in 2 (0.4%) and M. szulgai in 1 (0.2%). In 22 individuals clinically suspected of tubercular pleural effusion, pleural biopsy specimen gave higher isolation of mycobacteria (27.3%) as compared to isolations from pleural fluid specimens (9.1%).