Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis.

Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30µg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.

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.

Presumptive treatment for malaria is not justified in children receiving cancer chemotherapy.

BACKGROUND: Predominant etiologies of febrile neutropenia (FN) during the course of cancer chemotherapy include infections with bacteria, fungi, and viruses. Infection with malarial parasite is a possibility in regions that are endemic for malaria. Over-diagnosis and over-treatment of malaria is increasingly being recognized as a serious concern in malaria endemic regions. Aim was to determine the incidence of malarial infection in episodes of FN in children receiving chemotherapy for malignant disorders. METHODS: Children, with malignant disorders, on chemotherapy, who fulfilled the definition of FN were enrolled prospectively. Standard microscopy, quantitative buffy coat, and antigen detection (OptiMAL) were performed in each episode of FN. RESULTS: One hundred episodes of FN involving 82 children were investigated. The age ranged from 2 to 13 years (mean: 5.8 ± 2.8). Eighty-one episodes were in children with acute lymphoblastic leukemia, 15 in acute myeloid leukemia, and remaining 4 in other malignancies. Evidence for malaria was not found in any case by any of the three methods. CONCLUSIONS: Malaria was not found to be a causative agent for FN in children with various malignant disorders, in a region with low endemicity for malaria. Presumptive administration of antimalarials in children with FN is unjustified. Pediatric oncologists constantly face the challenge of managing febrile illnesses in immunocompromised patients. Those practicing in malaria endemic regions can effectively exploit diagnostic tools for malaria for a rational decision.

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: 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 non-cholera 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.

Specific antibody detection in serum, urine and saliva samples for the diagnosis of cystic echinococcosis.

Serum, saliva and urine samples of 25 clinically and radiologically diagnosed cystic echinoccosis (CE) patients, 25 clinically suspected cases of CE, 15 other parasitic disease controls and 25 healthy controls were evaluated for anti-hydatid antibody response by ELISA. The sensitivity of serum, saliva and urine was found to be 72, 56 and 84%, respectively, while specificity was 76% in all the samples. Urine showed significantly higher (p<0.05) sensitivity than that of saliva samples but not significantly higher (p>0.05) than that of serum samples. There was no significant difference in the immune response of patients with hepatic versus extrahepatic cysts and single versus multiple cysts. Thus, biological fluid like urine may be used as an alternative or as an adjunct to serum samples by virtue of its non-invasive, easy collection and similar sensitivity and specificity.

Antibody response to Toxoplasma gondii in saliva samples from human immunodeficiency virus-infected patients.

Toxoplasma gondii is an important opportunistic infection among human immunodeficiency virus (HIV)-infected patients as it causes fatal encephalitis. In the present study, antibody response to T. gondii is assessed in saliva samples from 100 HIV-seropositive patients and 25 HIV-negative healthy controls by indirect enzyme-linked immunosorbent assay (ELISA). Sensitivity and specificity for detection of IgG and IgM in saliva is calculated using a positive antibody response in serum samples (from an earlier study) as the gold standard. IgG and IgM antibodies were found in 20% and 25% patients, respectively. One control subject showed the presence of IgM antibody. Sensitivity for IgG and IgM antibodies was 64% and 81.25%, respectively, while specificity was 94.67% and 85.71%, respectively. This study indicates that saliva samples can be used as an alternative to serum samples to detect anti-toxoplasma antibodies, particularly IgM, for the diagnosis of toxoplasma encephalitis in HIV/acquired immune deficiency syndrome patients.

Antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus-infected patients.

Toxoplasma encephalitis in immunocompromised patients results from reactivation of previously acquired (latent) infection. The aim of the study is to assess the antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus (HIV)-infected patients to determine the best marker for early diagnosis of toxoplasmosis in such patients. Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG, IgM and IgA anti-toxoplasma antibodies and double-sandwich ELISA for toxoplasma antigen is carried out in serum samples collected from 100 HIV seropositive patients and 75 controls. Toxoplasma-specific IgG, IgM and IgA antibody response and antigenaemia were detected in 12%, 6%, 7% and 14% of HIV-infected patients, respectively. On retrospective analysis of 14 patients with antigenaemia only one had central nervous system (CNS) symptoms attributable to toxoplasma infection. In this patient, the CD4+ cell count was below 50/microL and none of the specific immunoglobulin isotype responses could be detected. The patient showed clinical improvement following specific chemotherapy for toxoplasmosis. In 25 HIV-negative and anti-toxoplasma IgG antibody-positive controls, IgM was detected in two (8%), IgA in five (20%) and antigenaemia in 10 (40%), while 50 HIV seronegative healthy controls were negative for both antigen and antibody responses. The study indicates that detection of toxoplasma antigen in addition to IgG antibody response may prove to be a useful indicator in the early diagnosis of reactivated toxoplasmosis in HIV/AIDS patients.

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.

Altered course of Plasmodium berghei infection by nifedipine treatment.

The effect of nifedipine (a calcium channel blocker) on the course of P. berghei infection was examined. It was observed that mice receiving a daily dose of 0.015 mg/kg of nifedipine had significantly shorter prepatent, patent and survival periods as compared to untreated P. berghei-infected animals (p < 0.001). This shows that the calcium channel blockers, in addition to possessing the property of reversing drug resistance during combined therapy with chloroquine, may also alter the pathophysiology of malaria infection. The decreased resistance of the host to the invading parasite suggests that the effect of CCB on the host-parasite interaction in human malaria needs to be investigated further before CCB can be used in combination with chloroquine for the treatment of chloroquine-resistant malaria or for chemoprophylaxis.

Generation of reactive oxygen species by blood monocytes during acute Plasmodium knowlesi infection in rhesus monkeys.

The status and kinetics of monocyte activation during acute P. knowlesi infection was investigated by latex-induced, luminol-dependent chemiluminescence (CL) response. The contribution of various reactive oxygen species (ROS) to CL response was estimated before infection and at peak parasitaemia (day 7 post infection) by using scavengers of ROS (benzoate, catalase and superoxide dismutase). The chemiluminescence index (CLI) was not found to be significantly different from controls on day 2 postinfection, but was significantly higher on days 5 and 7 postinfection. Hydroxyl radical (OH.) production was considerably elevated, whereas superoxide anion (O2-.) and hydrogen peroxide (H2O2) production dropped following infection. These changes in generation of ROS are discussed in relation to the progression of parasitaemia to high levels, immunopathology and immunosuppression during acute P. knowlesi infection.

Generation of reactive oxygen species by blood monocytes in human Plasmodium falciparum and P. vivax infections.

Generation of reactive oxygen radicals by peripheral blood monocytes was measured by luminol-dependent chemiluminescence in 23 P. vivax- and 7 P. falciparum-infected patients. The chemiluminescence index (CLI) was not found to be significantly higher in P. vivax-infected cases than in healthy controls. But in patients with P. falciparum infection, the CLI was significantly higher compared to controls as well as to P. vivax-infected patients. In two severe and complicated P. falciparum-infected cases, CLI was found to be higher than in mild cases. As immunosuppression is more marked in falciparum malaria than in vivax cases, the role of oxygen radical generation in immunopathology and causation of immunosuppression in falciparum malaria needs further investigation.

Plasmodium falciparum induced perturbations of the erythrocyte antioxidant system.

Erythrocyte antioxidants catalase, superoxide dismutase, reduced glutathione and glutathione peroxidase were studied in cells harbouring different growth stages of Plasmodium falciparum. Catalase and superoxide dismutase showed significant decrease during parasite maturation indicating hampered metabolism of hydrogen peroxide and superoxide anions. Glutathione peroxidase also exhibited a downward trend during the growth of P. falciparum, while there was a moderate accumulation of reduced glutathione. These findings suggest decreased utilization of the reduction potential in detoxification of reactive oxygen species. The fall in all three antioxidant enzymes studied was highly significant (P less than 0.001) in erythrocytes with mature stages of the parasite (trophozoites, schizonts). The increased vulnerability of erythrocytes to damage, which parallels the growth phases of the parasite emphasizes the need for early treatment of P. falciparum malaria to minimise red cell destruction and the resulting anaemia.

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%).

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.

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.

Effect of nifedipine on calcium status and chemiluminescence response of phagocytes during Plasmodium berghei infection in mice.

The macrophages and neutrophils from nifedipine-treated mice, both Plasmodium berghei-infected and uninfected, showed suppressed capacity to generate oxygen free radicals as compared with untreated controls. Nifedipine treatment did not affect resting state free calcium levels in these cells. But the rise in intracellular calcium levels of macrophages and neutrophils following P. berghei infection was significantly less (P < 0.05) in nifedipine-treated mice as compared with untreated groups at various parasitaemia levels. Probably this reflects a more potent effect of nifedipine on these cells in the depolarized state. Similarly, the rise in intracellular calcium levels of these cells following formyl-Met-Leu-Phe (fMLP) stimulation was also significantly less in nifedipine-treated groups than in untreated controls at different parasitaemia levels. A positive correlation between this fMLP-stimulated rise in calcium levels and the chemiluminescence response of macrophages and neutrophils was observed in nifedipine-treated and untreated groups at various parasitaemia levels. Thus the respiratory-burst responses of these cells during P. berghei infection depend on the calcium homeostasis in the cells. The disturbances of the calcium-regulating mechanisms by nifedipine treatment resulted in subnormal phagocytic cell responses which lead to more severe and rapidly fatal P. berghei infection in these animals.

Antigenaemia and antibody response to Leishmania donovani stage-specific antigens and rk39 antigen in human immunodeficiency virus-infected patients.

In order to define the possible markers for the early diagnosis of asymptomatic visceral leishmaniasis in human immunodeficiency virus (HIV)-infected individuals, the antigenaemia and antibody response to stage-specific Leishmania donovani and rk39 antigens is assessed by enzyme-linked immunosorbent assay (ELISA) and immunoreactivity to stage-specific antigens analysed by Western blot. Serum samples from two out of 100 HIV-infected individuals were found positive for antigenaemia, antibody response to stage-specific L. donovani antigens and rk39 antigen, and one sample was also positive for antigenaemia and antibody response to L. donovani antigens, while antibody detection to rk39 antigen was not carried on this sample. Additionally, one sample was found positive for amastigote antigenaemia and antibody response to amastigote antigen, while in this patient promastigote antigenaemia and antibody response to promastigote L. donovani and rk39 antigen could not be detected. One sample was found positive for antigenaemia, antibody response to amastigote antigen and negative for antibody response to promastigote antigen, while in this patient response to rk39 antigen was borderline. Although antibody response to rk39 antigen could be detected in 9/88 (10%) HIV-infected individuals, in six of these nine patients neither antigenaemia nor antibody response to stage-specific L. donovani antigens could be detected. All 10 confirmed visceral leishmaniasis and HIV-negative control patients had positive antigenaemia and antibody response to L. donovani amastigote and promastigote antigens, while all the normal healthy individuals were negative. The study indicated that detection of antibody response to rk39 antigen, amastigote antigenaemia and antibody response to amastigote antigen may prove to be better markers than detection of promastigote antigenaemia, antibody response to promastigote antigen and immunoblot reactivity.

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.