Taniawati Supali

Regulatory T cells in human geohelminth infection suppress immune responses to BCG and Plasmodium falciparum

Chronic helminth infections induce T‐cell hyporesponsiveness, which may affect immune responses to other pathogens or to vaccines. This study investigates the influence of Treg activity on proliferation and cytokine responses to BCG and Plasmodium falciparum‐parasitized RBC in Indonesian schoolchildren. Geohelminth‐infected childrens in vitro T‐cell proliferation to either BCG or pRBC was reduced compared to that of uninfected children. Although the frequency of CD4+CD25hiFOXP3+ T cells was similar regardless of infection status, the suppressive activity differed between geohelminth‐infected and geohelminth‐uninfected groups: Ag‐specific proliferative responses increased upon CD4+CD25hi T‐cell depletion in geohelminth‐infected subjects only. In addition, IFN‐γ production in response to both BCG and parasitized RBC was increased after removal of CD4+CD25hi T cells. These data demonstrate that geohelminth‐associated Treg influence immune responses to bystander Ag of mycobacteria and plasmodia. Geohelminth‐induced immune modulation may have important consequences for co‐endemic infections and vaccine trials.

Does treatment of intestinal helminth infections influence malaria? Background and methodology of a longitudinal study of clinical, parasitological and immunological parameters in Nangapanda, Flores, Indonesia (ImmunoSPIN Study)

BackgroundGiven that helminth infections are thought to have strong immunomodulatory activity, the question whether helminth infections might affect responses to malaria antigens needs to be addressed. Different cross-sectional studies using diverse methodologies have reported that helminth infections might either exacerbate or reduce the severity of malaria attacks. The same discrepancies have been reported for parasitemia.Methods/DesignTo determine the effect of geohelminth infections and their treatment on malaria infection and disease outcome, as well as on immunological parameters, the area of Nangapanda on Flores Island, Indonesia, where malaria and helminth parasites are co-endemic was selected for a longitudinal study. Here a Double-blind randomized trial will be performed, incorporating repeated treatment with albendazole (400 mg) or placebo at three monthly intervals. Household characteristic data, anthropometry, the presence of intestinal helminth and Plasmodium spp infections, and the incidence of malaria episodes are recorded. In vitro cultures of whole blood, stimulated with a number of antigens, mitogens and toll like receptor ligands provide relevant immunological parameters at baseline and following 1 and 2 years of treatment rounds. The primary outcome of the study is the prevalence of Plasmodium falciparum and P. vivax infection. The secondary outcome will be incidence and severity of malaria episodes detected via both passive and active follow-up. The tertiary outcome is the inflammatory cytokine profile in response to parasite antigens. The project also facilitates the transfer of state of the art methodologies and technologies, molecular diagnosis of parasitic diseases, immunology and epidemiology from Europe to Indonesia.DiscussionThe study will provide data on the effect of helminth infections on malaria. It will also give information on anthelminthic treatment efficacy and effectiveness and could help develop evidence-based policymaking.Trial registrationThis study was approved by The Ethical Committee of Faculty of Medicine, University of Indonesia, ref:194/PT02.FK/Etik/2006 and has been filed by ethics committee of the Leiden University Medical Center. Clinical trial number:ISRCTN83830814. The study is reported in accordance with the CONSORT guidelines for cluster-randomized studies.

Doxycycline Treatment of Brugia malayi-Infected Persons Reduces Microfilaremia and Adverse Reactions after Diethylcarbamazine and Albendazole Treatment

BACKGROUND The efficacy of doxycycline for treating the causal agent of human lymphatic filariasis, Brugia malayi, is unknown. Standard treatment with diethylcarbamazine-albendazole is associated with adverse reactions. We assessed whether doxycycline alone or in combination with diethylcarbamazine-albendazole would lead to sustained amicrofilaremia and reduced incidence of adverse reactions. METHODS A double-blind, randomized, placebo-controlled 6-week field trial of doxycycline treatment (100 mg/day) of 161 persons infected with B. malayi was conducted. Four months after receiving doxycycline (n=119) or placebo (n=42), participants received diethylcarbamazine (6 mg/kg) plus albendazole (400 mg) or a matching placebo. Adverse reactions were assessed 48 and 60 h after administration of diethylcarbamazine-albendazole. Treatment efficacy was evaluated at 2, 4, and 12 months after the initial doxycycline treatment. RESULTS Four months after beginning doxycycline treatment, Wolbachia loads were reduced by 98%. Doxycycline treatment reduced the prevalence of microfilaremia at 2, 4, and 12 months of follow-up (P<.001 for all time points). At the 1-year follow-up, prevalence was reduced by 77% and 87.5% in patients receiving doxycycline alone or doxycycline plus diethylcarbamazine-albendazole, respectively. In contrast, the reduction of microfilaremia in the group receiving placebo doxycycline plus diethylcarbamazine-albendazole was merely 26.7%. Adverse reactions were lowest in the group receiving doxycycline plus placebo diethylcarbamazine-albendazole and highest in the group receiving placebo doxycycline plus diethylcarbamazine-albendazole. The proportion of persons with high fever and severe adverse reactions was significantly reduced in the group treated with doxycycline plus diethylcarbamazine-albendazole. CONCLUSIONS A 6-week course of doxycycline, either alone or in combination with diethylcarbamazine-albendazole, leads to a decrease in microfilaremia and reduces adverse reactions to antifilarial treatment in B. malayi-infected persons.

Polyparasitism and its impact on the immune system.

Parasitic infections are common in many tropical and sub-tropical regions of the world and concomitant infection, polyparasitism, is the rule rather than the exception in such areas. At the immunological level, different parasites induce quite different responses characterised, for example, by protozoa that polarise responses towards Th1, whilst helminths are strong Th2 and regulatory T cell inducers. The question of how the co-existence of such parasites within the same host might influence the immunological responses to each species and, more importantly, whether such interactions affect resistance, susceptibility or clinical outcome, needs to be addressed in well-designed studies of sufficient power. The current paper discusses what we know as well as the gaps in our knowledge of polyparasitism.

Helminth genome analysis: The current status of the filarial and schistosome genome projects

Genome projects for the parasitic helminths Brugia malayi (a representative filarial nematode) and Schistosoma were initiated in 1995 by the World Health Organization with the ultimate objectives of identifying new vaccine candidates and drug targets and of developing low resolution genome maps. Because no genetic maps are available, and very few genes have been characterized from either parasite group, the first goal of both Initiatives has been to catalogue new genes for future placement on chromosome and physical maps. These genes have been identified by the expressed sequence tag (EST) approach, utilising cDNA libraries constructed from diverse life cycle stages. To date, the Initiatives have deposited over 16,000 Brugia ESTs and nearly 8000 Schistosoma ESTs in Genbanks dbEST database, corresponding to 6000 and over 3600 genes respectively (33% of Brugias estimated gene compliment, 18-24% of that of Schistosoma). Large fragment, genomic libraries have been constructed in BAC and YAC vectors for studies of genomic organization and for physical and chromosome mapping, and public, hypertext genomic databases have been established to facilitate data access. We present a summary of progress within the helminth genome initiatives and give several examples of important gene discoveries and future applications of these data.

PCR-based detection and identification of the filarial parasite Brugia timori from Alor Island, Indonesia.

Brugia timori is widely distributed on Alor Island, Indonesia, where it causes a high degree of morbidity. The HhaI tandem repeat of B. timori was found to be identical to that of B. malayi, for which sensitive PCR-based assays have already been developed. Using one of these assays, a single microfilaria (mf) of B. timori, present in a spot of dry blood on filter paper, could be detected. The assay was equally sensitive in the detection of B. timori and B. malayi. When the collected mosquitoes were pooled according to species and tested with the assay, 39 (64%) of the 61 Anopheles barbirostris pools (containing a total of 642 mosquitoes) were positive. As none of the 33 Culex pools tested (which contained 624 mosquitoes) gave a positive result, and An. barbirostris is the only Anopheles species commonly caught on human bait in Alor, An. barbirostris is assumed to be the main and perhaps only local vector. Brugia timori could be differentiated from B. malayi by restriction-endonuclease digestion of the PCR-amplified mitochondrial cytochrome oxidase subunit 2. A few distinct nucleotide exchanges were also found in the second internal transcribed ribosomal spacer of the filariae, and in the 16S rDNA and FTSZ gene of their Wolbachia endobacteria. The results show that B. timori can be effectively detected using the PCR-based assay developed for B. malayi and can then be differentiated from B. malayi by other molecular markers. PCR-based techniques targeting the HhaI repeat can therefore be employed for monitoring B. timori in the framework of the Global Programme to Eliminate Lymphatic Filariasis.

Detection of Brugia Parasite DNA in Human Blood by Real-Time PCR

ABSTRACT Brugian filariasis (caused by the nematodes Brugia malayi and B. timori) is an important cause of disability in Southeast Asia. Improved diagnostic tests are needed for filariasis elimination programs (to identify areas of endemicity and to monitor progress) and for diagnosis of the disease in infected individuals. We have developed and evaluated two real-time PCR assays for detecting Brugia DNA in human blood and compared the results of these assays to those of “gold standard” assays. One assay uses a TaqMan probe (TaqM) to amplifiy a 320-bp “HhaI repeat” DNA sequence. The other assay uses a minor groove binding probe (MGB) and modified nucleotides in primers (Eclipse MGB) to amplify a 120-bp fragment of the HhaI repeat. This assay detects 22 copies of the target sequence, and it is more sensitive than the TaqM assay. Both assays were evaluated with human blood samples from two different areas of endemicity. The MGB assay was as sensitive as membrane filtration and microscopy for the detection of B. malayi infection in 57 blood samples recovered at night from patients in Sulawesi, Indonesia. The MGB assay also detected parasite DNA in 17 of 31 (55%) of microfilaria-negative day blood samples from these subjects. This test was more sensitive than the conventional and the TaqM PCRs (and was almost as sensitive as night blood membrane filtration) for the detection of infection in 52 blood samples recovered at night from individuals in an area of B. timori endemicity on Alor Island, Indonesia, where microfilaria-positive individuals had low densities after mass treatment. Thus, the Eclipse MGB real-time PCR assay is a sensitive means of detecting Brugia parasite DNA in human blood.

Transmission assessment surveys (TAS) to define endpoints for lymphatic filariasis mass drug administration: a multicenter evaluation.

Background Lymphatic filariasis (LF) is targeted for global elimination through treatment of entire at-risk populations with repeated annual mass drug administration (MDA). Essential for program success is defining and confirming the appropriate endpoint for MDA when transmission is presumed to have reached a level low enough that it cannot be sustained even in the absence of drug intervention. Guidelines advanced by WHO call for a transmission assessment survey (TAS) to determine if MDA can be stopped within an LF evaluation unit (EU) after at least five effective rounds of annual treatment. To test the value and practicality of these guidelines, a multicenter operational research trial was undertaken in 11 countries covering various geographic and epidemiological settings. Methodology The TAS was conducted twice in each EU with TAS-1 and TAS-2 approximately 24 months apart. Lot quality assurance sampling (LQAS) formed the basis of the TAS survey design but specific EU characteristics defined the survey site (school or community), eligible population (6–7 year olds or 1st–2nd graders), survey type (systematic or cluster-sampling), target sample size, and critical cutoff (a statistically powered threshold below which transmission is expected to be no longer sustainable). The primary diagnostic tools were the immunochromatographic (ICT) test for W. bancrofti EUs and the BmR1 test (Brugia Rapid or PanLF) for Brugia spp. EUs. Principal Findings/Conclusions In 10 of 11 EUs, the number of TAS-1 positive cases was below the critical cutoff, indicating that MDA could be stopped. The same results were found in the follow-up TAS-2, therefore, confirming the previous decision outcome. Sample sizes were highly sex and age-representative and closely matched the target value after factoring in estimates of non-participation. The TAS was determined to be a practical and effective evaluation tool for stopping MDA although its validity for longer-term post-MDA surveillance requires further investigation.

Regulatory T Cells in Human Lymphatic Filariasis: Stronger Functional Activity in Microfilaremics

Infection with filarial parasites is associated with T cell hyporesponsiveness, which is thought to be partly mediated by their ability to induce regulatory T cells (Tregs) during human infections. This study investigates the functional capacity of Tregs from different groups of filarial patients to suppress filaria-specific immune responses during human filariasis. Microfilaremic (MF), chronic pathology (CP) and uninfected endemic normal (EN) individuals were selected in an area endemic for Brugia timori in Flores island, Indonesia. PBMC were isolated, CD4CD25hi cells were magnetically depleted and in vitro cytokine production and proliferation in response to B. malayi adult worm antigen (BmA) were determined in total and Treg-depleted PBMC. In MF subjects BmA-specific T and B lymphocyte proliferation as well as IFN-gamma, IL-13 and IL-17 responses were lower compared to EN and CP groups. Depletion of Tregs restored T cell as well as B cell proliferation in MF-positives, while proliferative responses in the other groups were not enhanced. BmA-induced IL-13 production was increased after Treg removal in MF-positives only. Thus, filaria-associated Tregs were demonstrated to be functional in suppressing proliferation and possibly Th2 cytokine responses to BmA. These suppressive effects were only observed in the MF group and not in EN or CP. These findings may be important when considering strategies for filarial treatment and the targeted prevention of filaria-induced lymphedema.

A Longitudinal Study of BCG Vaccination in Early Childhood: The Development of Innate and Adaptive Immune Responses

BCG vaccine drives a strong T helper 1 cellular immunity which is essential for the protection against mycobacteria, however recent studies suggest that BCG vaccination can have non-specific beneficial effects unrelated to tuberculosis. In the present cohort study the development of cytokine profiles following BCG vaccination was investigated. Immune responses to PPD were assessed before vaccination and at ages of 5 months, 1 year, and 2 years, followed by BCG scar measurement at 4 years of age. BCG was shown to induce both Th1 and Th2 type responses against PPD at about 5 months of age after vaccination, and while Th1 response was sustained, Th2 responses declined over time. However, BCG scar size was strongly correlated with Th2 responses to PPD at 5 months of age. Importantly, we observed no clear effects of BCG vaccination on innate immune responses in terms of early IL-10 or TNF-α production whereas some alterations in general adaptive immune responses to PHA were observed.