Nancy Malla

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.

In vitro activity of antiamoebic drugs against clinical isolates of Entamoeba histolytica and Entamoeba dispar

BackgroundAmoebiasis is a major public health problem in tropical and subtropical countries. Although a number of antiamoebic agents are used for its treatment, yet the susceptibility data on clinical isolates of Entamoeba histolytica and Entamoeba dispar are not available. Therefore, the present study was aimed to assess the in vitro susceptibility of clinical isolates of E. histolytica and E. dispar to metronidazole, chloroquine, emetine and tinidazole.MethodsA total of 45 clinical isolates (15 E. histolytica and 30 E. dispar) were maintained in polyxenic cultures followed by monoxenic cultures. In vitro drug sensitivity (IC50) of clinical isolates and standard reference strain of E. histolytica (HM1: IMSS) was assessed by nitro blue tetrazolium (NBT) reduction assay after exposure to various concentrations of each drug.ResultsThe results showed that all clinical isolates had a higher IC50 compared to reference strain to all the four drugs. E. histolytica isolates appeared to be more susceptible [IC50 (μm) 13.2,26.3,31.2 and 12.4] compared to E. dispar isolates [IC50(μm) 15.6,28.9,32.8 and 13.2] and the reference strain of E. histolytica [IC50 (μm) 9.5, 15.5, 29.9 and 10.2] to the metronidazole, chloroquine, emetine and tinidazole respectively.ConclusionsThe results indicate that till date, Entamoeba isolates in India do not seem to be resistant to the commonly used antiamoebic drugs.

Immunogenicity and efficacy of single antigen Gp63, polytope and polytopeHSP70 DNA vaccines against visceral Leishmaniasis in experimental mouse model.

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-γ over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.

Molecular characterization of Echinococcus granulosus cysts in north Indian patients: identification of G1, G3, G5 and G6 genotypes.

Background Cystic echinococcosis (CE) caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s) of the parasite responsible for human hydatidosis in North India. Methodology/Principal Findings To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE) patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1) sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32) were found to be infected with hydatid cyst of either G3 (53.1%) or G1 (40.62%) genotype and one each of G5 (cattle strain) and G6 (camel strain) genotype. Conclusions/Significance These findings demonstrate the zoonotic potential of G1 (sheep strain) and G3 (buffalo strain) genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain) in an Asian country and presence of G6 genotype (camel strain) in India. The results may have important implications in the planning of control strategies for human hydatidosis.

Genetic diversity of Cryptosporidium isolates from patients in North India

BACKGROUND Cryptosporidiosis is a significant cause of diarrheal illness in both immunocompetent and immunocompromised populations. Cryptosporidium species infect a wide range of hosts including humans. Different species are morphologically indistinguishable, and molecular techniques have become the key to detection and source tracking. The present study was designed to study the genetic diversity of human Cryptosporidium isolates in North India. METHODS Cryptosporidium oocysts were detected in stool samples by special staining of fecal smears. DNA was extracted with a Qiagen kit and all samples were genotyped by small subunit ribosomal ribonucleic acid (SSU rRNA)-based nested PCR-restriction fragment length polymorphism (RFLP) tool using enzymes SspI and VspI. Cryptosporidium hominis and Cryptosporidium parvum isolates were subtyped by sequence analysis of the nested PCR amplified gp60 gene. RESULTS Fifty-three fecal samples were found to be positive for Cryptosporidium oocysts. RFLP analysis revealed 39 isolates as C. hominis and 13 isolates of C. parvum; one sample failed amplification. gp60-based sequencing of C. hominis and C. parvum divided them into eight subgenotype families and 17 subtypes. gp60-based sequencing identified seven cases of mixed infection with C. hominis and C. parvum/Cryptosporidium meleagridis and showed the presence of C. meleagridis in six HIV-positive patients that were indistinguishable in RFLP. CONCLUSIONS Cryptosporidium isolates obtained in the present study from patients in North India belonged to three species, eight subgenotype families, and 17 subtypes. The existence of many Cryptosporidium species, subgenotypes, and subtypes along with mixed infections reveals the complexity of Cryptosporidium transmission; this heterogeneity indicates stable cryptosporidiosis transmission in North India. The results may have further implications in understanding the epidemiology and control of this infection.

Specific IgA response, T-cell subtype and cytokine profile in experimental intravaginal trichomoniasis.

Abstract. Trichomoniasis caused by Trichomonas vaginalis may lead to either a complete absence of symptoms or to severe inflammatory manifestations in infected women. Studies of the role of immune responses in the pathogenesis and varied symptomatology of this disease are lacking. Mice may prove useful as an experimental model for intravaginal trichomoniasis in developing an understanding of the role of local immune responses in the pathogenesis and varied symptomatology of this disease. The present study reports the levels of anti-Trichomonas IgA antibodies in serum and vaginal washes, and T-cell subtype and cytokine profile in vaginal cervical tissues of mice infected intravaginally with T. vaginalis isolates from 15 symptomatic and 15 asymptomatic women. It also correlates the responses with symptomatology of the patients. Successful intravaginal infection was established by inoculating T. vaginalis in BALB/c mice preinoculated with Lactobacillus acidophilus and pretreated with oestradiol. A significant increase in specific IgA antibody levels was detected with enzyme-linked immunosorbent assay in vaginal secretions and serum samples collected on the 7th post-infection day from animals infected with isolates from asymptomatic women when compared with mice infected with isolates from symptomatic women. T-cell subset analysis showed significant differences, with increased CD4+ T-cell count in animals infected with isolates from asymptomatic women compared with animals infected using isolates from symptomatic women. No difference in CD8+ T cells was observed between the two groups. Cytokine profile revealed significantly higher (P<0.001) production of γ-IFN and IL-2 in mice infected with asymptomatic isolates compared with animals infected with symptomatic isolates, using T. vaginalis crude antigen extract and nonspecific mitogen (ConA) as stimulants for vaginal cervical lymphocytes. However, no difference in IL-4 levels was observed in the two groups of animals. In contrast, significant increase in tumour necrosis factor (TNF-α) levels was observed in animals infected with asymptomatic isolates compared with those infected with isolates from symptomatic women and controls, thereby indicating that TNF-α may play an important role in the inflammatory response to trichomoniasis. The study further suggests that specific IgA antibodies might help to protect asymptomatic individuals from severe infection and T-lymphocytes may play an important function in the eradication of the parasite. The cytokine profile indicated the involvement of Th-1 like responses in mice infected with asymptomatic isolates, compared with those infected with symptomatic isolates.

Evaluation of a 200-kDa amastigote-specific antigen of L. donovani by enzyme-linked immunosorbent assay (ELISA) for the diagnosis of visceral leishmaniasis

A purified 200-kDa antigenic fraction from Leishmania donovani axenic amastigotes was evaluated by ELISA for the detection of antibody response in visceral leishmaniasis (VL) patients, post kala-azar dermal leishmaniasis (PKDL) patients and controls, for the diagnosis of visceral leishmaniasis. A positive antibody response to the 200-kDa amastigote fraction and to Leishmania amastigote soluble antigen (LASA) was found in 29 (96.6%) and 30 ((100%) confirmed VL patients, respectively, by the use of ELISA. However, only 1 (10%) out of 10 PKDL patients had detectable antibody response to 200-kDa fraction while all the 10 (100%) PKDL patients exhibited an immune response to LASA. Therefore, use of the 200-kDa antigenic fraction for the detection of antibody response in an ELISA follow-up (post treatment) of VL patients may have prognostic significance, and it may also be useful for differentiating active VL and PKDL.

Cellular immune responses in human neurocysticercosis

Abstract Studies on the role of cell-mediated immune responses in human neurocysticercosis (NCC) are lacking. Various cell-mediated immune responses such as lymphocyte subpopulation, lymphocyte transformation to cysticercus antigens and cytokine profile were carried out in NCC patients. Lymphocyte transformation assays using larval antigens showed significantly higher 3H-thymidine uptake. Immunophenotyping analysis showed an insignificant increase in B cells and a decrease in total T cells. However, there was a significant decrease (P < 0.05) in CD8+ T cells whereas there was no change in other cells like CD4+, HLA-DR+ and CD16+/CD56+. Cytokine profile revealed significantly higher (P < 0.01) production of Th1 cytokines (γ-IFN and IL-2) using cysticercal antigens as stimulants for peripheral blood mononuclear cells, while there was no difference in IL-4 levels between NCC patients and healthy controls. The cytokine profile indicated the involvement of Th-1-like responses in NCC patients.

Kinetics of immunoglobulin G, M, A and IgG subclass responses in experimental intravaginal trichomoniasis: prominence of IgG1 response

Trichomoniasis, the most common nonviral sexually transmitted disease, is caused by infection with the protist Trichomonas vaginalis. The clinical spectrum varies from an asymptomatic state to mild, moderate or severe symptoms. However, the exact factors leading to varied symptomatology have not been well elucidated. The mouse is a useful experimental model for intravaginal trichomoniasis, for understanding the role of local immune responses in the pathogenesis and varied symptomatology of this disease. The present study reports anti‐Trichomonas IgG, IgM, IgA and IgG subclass antibody responses on different post‐infection days (3rd, 7th, 14th, 21st and 28th) in serum and vaginal washes of mice infected intravaginally with T. vaginalis isolates from 15 symptomatic and 15 asymptomatic women. Successful intravaginal infection was established by inoculating T. vaginalis in BALB/c mice pre‐inoculated with Lactobacillus acidophilus and pretreated with oestradiol. A significant increase in parasite load was observed on the 14th post‐infection day (p.i.d.) in mice inoculated with T. vaginalis isolates from symptomatic women as compared to asymptomatic women (P < 0·001), followed by reduction until the 28th p.i.d. (P < 0·05). A significant increase in specific IgG (P < 0·001) and in particular IgG1 (P < 0·001) and IgM (P < 0·01) responses was observed on the 14th p.i.d. in serum and vaginal washes of mice infected with T. vaginalis isolates from symptomatic women as compared to asymptomatic women. Significant increases in specific IgG, IgM and IgG1 responses was observed on the 14th p.i.d. in serum samples as compared with vaginal washes of mice infected with T. vaginalis isolates from symptomatic and asymptomatic women (P < 0·01), whereas no significant difference was observed in IgA, IgG2a, IgG2b and IgG3 antibody responses. The study indicates that specific IgG, particularly IgG1 and IgM, may be playing a role in establishing symptomatic infection.

Evaluation of Ziehl-Neelsen staining, auramine phenol staining, antigen detection enzyme linked immunosorbent assay and polymerase chain reaction, for the diagnosis of intestinal cryptosporidiosis

Background and Objectives: Cryptosporidiosis is a very important opportunistic infection and is responsible for significant morbidity and mortality in HIV/AIDS patients. The objective of this study is to evaluate Ziehl-Neelsen staining, auramine phenol staining, antigen detection enzyme linked immunosorbent assay and polymerase chain reaction, for the diagnosis of intestinal cryptosporidiosis. Materials and Methods: The study was designed to determine the efficacy of modified Ziehl-Neelsen (ZN), Auramine-Phenol (AP) staining, antigen detection enzyme linked immunosorbent assay (ELISA) and nested polymerase chain reaction (PCR) for detection of cryptosporidia in 671 HIV-seropositive patients, 353 HIV-seronegative patients including 198 children with diarrhea and 50 apparently healthy adults. Results: Cryptosporidium was detected in 26 (3.9%), 37 (5.5%), 32 (4.8%) and 40 (6%) HIV-seropositive and 8 (2.3%), 10 (2.9%), 9 (2.6%) and 9 (2.6%) HIV-seronegative patients by ZN staining, AP staining, antigen detection ELISA and PCR, respectively. None of the healthy controls were infected with Cryptosporidium. Based on criteria of ′true positive′ samples, i.e. positive by any two of the four techniques out of ZN, AP, antigen detection ELISA and PCR, sensitivity of ZN and ELISA was 79.06% and 95.35% respectively. AP and PCR were found to be 100% sensitive. Specificity of ZN and ELISA was 100% while specificity of AP and PCR was 99.59% and 99.39% respectively. Conclusions: Auramine phenol staining is a rapid, sensitive and specific technique for diagnosis of intestinal cryptosporidiosis.