Stage-specific antibody response against two larval stages of Brugia malayi in different clinical spectra of brugian filariasis.

CONTEXT: T-cell hypo-responsiveness in microfilaria (Mf) carriers against the microfilarial stage antigen of Brugia malayi has been described, but no study has been carried out to assess antibody dynamics against stage-specific antigens. AIM: The work was carried out with the aim to assess stage-specific antibody responses against L3 and microfilarial stage antigens in brugian filariasis in an endemic area. SETTING AND DESIGN: Patients with different clinical spectra of brugian filariasis were recruited to evaluate antibody responses to brugian antigens. SUBJECTS AND METHODS: Serum samples were collected from patients with different clinical spectra and antibody response was evaluated for total immunoglobulin G (IgG), IgG isotypes (IgG1, IgG2, IgG3, IgG4) and immunoglobulin E (IgE) response to L3 and microfilarial stage by enzyme-linked immunosorbent assay. STATISTICAL ANALYSIS: Paired t-test and one-way analysis of variance were carried out to analyze the data. RESULTS: L3 and microfilarial stage antigens showed almost similar antibody responses in adenolymphangitis (ADL) and chronic pathology (CP) patients, however, diminished antibody response was observed with Mf stage antigen, especially with microfilaraemia. ADL patients had minimum antibody levels of all isotypes except IgG2 on day 0 which showed an increase subsequently, indicating suppression of antibody response during filarial fever. CP patients showed increase in IgE and decrease in IgG4 antibodies on day 365 indicating that these differences may be due to recent conversion into CP. CONCLUSION: A prominent hyporesponsiveness in microfilaraemic individuals against microfilarial stage, but not against the L3 stage of the same parasite was observed, concluding stage-specificity in humoral immune response in brugian filariasis.

Circulating filarial antigen detection in brugian filariasis.

Human lymphatic filariasis (LF) is a major cause of disability globally. The success of global elimination programmes for LF depends upon effectiveness of tools for diagnosis and treatment. In this study on stage-specific antigen detection in brugian filariasis, L3, adult worm (AW) and microfilarial antigenaemia were detected in around 90-95% of microfilariae carriers (MF group), 50-70% of adenolymphangitis (ADL) patients, 10-25% of chronic pathology (CP) patients and 10-15% of endemic normal (EN) controls. The sensitivity of the circulating filarial antigen (CFA) detection in serum samples from MF group was up to 95%. In sera from ADL patients, unexpectedly, less antigen reactivity was observed. In CP group all the CFA positive individuals were from CP grade I and II only and none from grade III or IV, suggesting that with chronicity the AWs lose fecundity and start to disintegrate and die. Amongst EN subject, 10-15% had CFA indicating that few of them harbour filarial AWs, thus they might not be truly immune as has been conventionally believed. The specificity for antigen detection was 100% when tested with sera from various other protozoan and non-filarial helminthic infections.

Utility of specific IgG4 response in saliva and serum samples for the diagnosis and follow up of human neurocysticercosis.

This study was aimed to analyze the Ig subtypes and IgG1 and IgG4 subclass responses to crude soluble extract (CSE) antigen and Ag B of Cysticercus cellulosae in pre and post treatment (PT) saliva and serum samples for the diagnosis and follow-up of neurocysticercosis (NCC) patients. Saliva and serum samples collected from 55 patients (15 highly suggestive of NCC clinically and radiologically, 10 hydatidosis, 10 other helminthic infections, 10 tubercular meningitis, 10 neurological disorders other than NCC), 15 normal healthy subjects and 10 NCC patients at 1, 3 and 6 months following albendazole therapy were analyzed for specific IgG, IgG1, IgG4, IgM and IgA antibody responses by ELISA. With the use of CSE Ag, the rank orders in saliva for sensitivity was IgG (71.4%) > IgG1 (68.2%) > IgG4 (65.2%) > IgM (57.7%) > IgA (55.5%) and specificity IgG1 = IgA (93.2%) > IgG = IgG4 = IgM (91.6%) while in serum, sensitivity was IgG (78.9%) > IgG1 (71.4%) > IgG4 (68.2%) > IgA (65.2%) > IgM (62.5%) and specificity IgG1 = IgG4 (90.2%) > IgA (85.9%) > IgG (83.3%) > IgM (82.1%). With the use of Ag B in saliva, the sensitivity was IgG (65.2%) > IgG1 = IgG4 (62.5%) > IgM = IgA (55.5%) and specificity with the use of saliva was IgG1 = IgG4 = IgM (94.8%) > IgG (93.2%) > IgA (91.6%) while with serum the sensitivity was IgG = IgG1 (68.2%) > IgG4 (65.2%) > IgA (62.5%) > IgM (57.7%) and specificity was IgG1 (93.2%) > IgG4 = IgM (91.6%) > IgA (90.2%) > IgG (87.3%). Comparative analysis of antibody responses in patients with single Vs multiple CT scan lesions indicated higher sensitivity in multiple lesion patients. Antibody responses in PT samples indicated that the undetectable IgG4, IgM and IgA responses in saliva samples correlated well with the CT scan reports while in serum samples, responses persisted longer. In conclusion, this study indicated that due to the lower sensitivity of IgM and IgA responses in pretreatment samples, detection of IgG4 subclass in saliva to either CSE Ag or AgB may serve better marker in the NCC follow-up.

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

BACKGROUND: Amoebiasis 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. METHODS: A 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. RESULTS: The 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 (microm) 13.2,26.3,31.2 and 12.4] compared to E. dispar isolates [IC50(microm) 15.6,28.9,32.8 and 13.2] and the reference strain of E. histolytica [IC50 (microm) 9.5, 15.5, 29.9 and 10.2] to the metronidazole, chloroquine, emetine and tinidazole respectively. CONCLUSIONS: The results indicate that till date, Entamoeba isolates in India do not seem to be resistant to the commonly used antiamoebic drugs.

Flowcytometric assessment of the effect of drugs on Giardia lamblia trophozoites in vitro.

The effect of albendazole, vincristine and metronidazole on the adherence, morphology, cell cycle and viability of Giardia lamblia trophozoites was evaluated. Albendazole rendered the trophozoites unable to attach which is a prerequisite for establishment of Giardia infection. The adherence assay showed that albendazole caused the detachment of trophozoites at a significantly lower concentration (0.1 microg/ml) as compared to metronidazole and vincristine (40 and 60 microg/ml, respectively) in the time period of 9 h. However, at the higher concentration (10 microg/ml) albendazole caused the similar effect within the time period of 3 h. The microscopic studies revealed that albendazole brought about gross morphological changes in G. lamblia, i.e., rounding of trophozoites and disruption of ventral adhesive disc along with vacuolation in cytoplasm. Metronidazole caused the ballooning of cytoplasm and induced the most lethal effect on trophozoites (8.17% viability) as compared to vincristine and albendazole (72.13 and 65.49% viability) as shown by flowcytometric analysis. Further, the flowcytometric findings indicated that the three drugs led to the arrest of growth of trophozoites at different stages of cell cycle. Albendazole and vincristine interfered with the formation of spindle/basal body microtubules thereby arresting the cell cycle in G2 + M phase whereas metronidazole arrested the growth in S-phase by inhibiting the DNA segregation and cell division. Hence, flowcytometry has been usefully employed to study the effect of drugs on viability and DNA of G. lamblia trophozoites in the present study.

Intestinal parasites and intra familial incidence in a low socio-economic area of Chandigarh (North India).

A total of 550 stool samples were collected from a low socio economic population of Chandigarh (North India) and examined macroscopically and microscopically, to determine the prevalence of intestinal parasitic infections and their familial incidence. The overall prevalence rate was 19.3%. Ascaris lumbricoides and Giardia lamblia were the commonest, affecting 51 (9.3%) and 33 (6.0%), respectively. In 17 (22.7%) families the same parasite was observed to infect multiple family members, which included A.lumbricoides (in 9 families), G. lamblia (in 7 families) and H. nana (in 1 family). The results of present study indicate that there is a high prevalence of parasitic infection in the community where personal hygiene and sanitary conditions are poor and may be one of the contributing factors for transmission within the families. Intervention strategies including health education program should be designed and implemented to control parasitic infections.

Comparative evaluation of hexokinase isoenzyme typing and antigen detection for differentiation of Entamoeba histolytica and Entamoeba dispar.

Entamoeba histolytica, the causative organism of invasive amebiasis is a potential pathogen, while asymptomatic infection is caused by E. dispar. Differentiation of the species is not possible on the basis of morphological characters by microscopic examination. In the present study an attempt has been made to differentiate E. histolytica from E. dispar in 45 isolates obtained from culture and direct stool samples respectively on the basis of hexokinase isoenzyme analysis and Tech Lab ELISA. A 100% correlation was found between these two techniques. However, Tech Lab E. histolytica antigen detection test was found to be both rapid and technically simple. Its use in diagnosis and epidemiological studies is recommended.

DNA polymorphism in north indian isolates of Entamoeba histolytica detected by PCR fingerprinting.

Entamoeba histolytica infection in humans is still one of the leading causes of parasitic diseases among the developing countries, including India. It therefore becomes extremely important to characterize this parasite with the aim of preventing and controlling the amoebiasis which it causes. The present study describes for the first time the ability of random amplified polymorphic DNA (RAPD)-PCR fingerprinting strategies to subtype amoebae and to detect considerable genetic variability present among the various North Indian isolates of E. histolytica studied. The number of rapdemes generated by the application of arbitrary primers in RAPD-PCR showed a significant variation in these isolates, both in the size and number of bands, thus revealing the presence of considerable genetic polymorphism in them. Hence, this rapid and easy method could suitably be employed in carrying out significant molecular epidemiological studies and also in the large-scale epidemiological typing of this parasite.

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting.

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.