Use of Antibodies in Lymphocyte Secretions for Detection of Subclinical Tuberculosis Infection in Asymptomatic Contacts

We have previously demonstrated that Mycobacterium bovis BCG-specific immunoglobulin G antibodies in lymphocyte secretions (ALS) can be employed as a marker for active tuberculosis (TB). We aimed to determine whether the ALS method allows detection of subclinical TB infection in asymptomatic individuals. A prospective study of family contacts (FCs) of patients with active TB and healthy controls was performed. Thirteen of 42 FCs had high ALS responses, including 6 FCs who subsequently developed active TB. No correlation was observed between the tuberculin skin test and the ALS responses in the FCs (r = 0.1, P = 0.23). Among patients with active TB, BCG-specific ALS responses steadily declined from the time of diagnosis through 6 months following antimycobacterial chemotherapy (P = 0.001). The ALS assay enabled detection of infection in exposed symptom-free contacts, who are at greater risk for developing active TB. The method may also allow discrimination between effective treatment of active infection and suboptimal response to therapy.     

Antibody-Secreting Cell Responses after Vibrio cholerae O1 Infection and Oral Cholera Vaccination in Adults in Bangladesh

Infection with Vibrio cholerae and oral cholera vaccines (OCVs) induce transient circulating plasmablast responses that peak within approximately 7 days after infection or vaccination. We previously demonstrated that plasmablast responses strongly correlate with subsequent levels of V. cholerae-specific duodenal antibodies up to 6 months after V. cholerae infection. Hence, plasmablast responses provide an early window into the immunologic memory at the mucosal surface. In this study, we characterized plasmablast responses following V. cholerae infection using a flow cytometrically defined population and compared V. cholerae-specific responses in adult patients with V. cholerae O1 infection and vaccinees who received the OCV Dukoral (Crucell Vaccines Canada). Among flow cytometrically sorted populations of gut-homing plasmablasts, almost 50% of the cells recognized either cholera toxin B subunit (CtxB) or V. cholerae O1 lipopolysaccharide (LPS). Using a traditional enzyme-linked immunosorbent spot assay (ELISPOT), we found that infection with V. cholerae O1 and OCVs induce similar responses to the protein antigen CtxB, but responses to LPS were diminished after OCV compared to those after natural V. cholerae infection. A second dose of OCV on day 14 failed to boost circulating V. cholerae-specific plasmablast responses in Bangladeshi adults. Our results differ from those in studies from areas where cholera is not endemic, in which a second vaccination on day 14 significantly boosts plasmablast responses. Given these results, it is likely that the optimal boosting strategies for OCVs differ significantly between areas where V. cholerae infection is endemic and those where it is not.     

Impact of sampling time deviations on the prediction of the area under the curve using regression limited sampling strategies

The regression limited sampling strategy approach (R‐LSS), which is based on a small number of blood samples drawn at selected time points, has been used as an alternative method for the estimation of the area under the concentration–time curve (AUC). However, deviations from planned sampling times may affect the performance of R‐LSS, influencing related therapeutic decisions and outcomes. The aim of this study was to investigate the impact of different sampling time deviation (STD) scenarios on the estimation of AUC by the R‐LSS using a simulation approach. Three types of scenarios were considered going from the simplest case of fixed deviations, to random deviations and then to a more realistic case where deviations of mixed nature can occur. In addition, the sensitivity of the R‐LSS to STD in each involved sampling point was evaluated. A significant impact of STD on the performance of R‐LSS was demonstrated. The tolerance of R‐LSS to STD was found to depend not only on the number of sampling points but more importantly on the duration of the sampling process. Sensitivity analysis showed that sampling points at which rapid concentration changes occur were relatively more critical for AUC prediction by R‐LSS. As a practical approach, nomograms were proposed, where the expected predictive performance of R‐LSS was provided as a function of STD information. The investigation of STD impact on the predictive performance of R‐LSS is a critical element and should be routinely performed to guide R‐LSS selection and use. Copyright © 2015 John Wiley & Sons, Ltd.     

Immune Responses to the O-Specific Polysaccharide Antigen in Children Who Received a Killed Oral Cholera Vaccine Compared to Responses following Natural Cholera Infection in Bangladesh

Current oral cholera vaccines induce lower levels of protective efficacy and shorter durations of protection in young children than in adults. Immunity against cholera is serogroup specific, and immune responses to Vibrio cholerae lipopolysaccharide (LPS), the antigen that mediates serogroup-specific responses, are associated with protection against disease. Despite this, responses against V. cholerae O-specific polysaccharide (OSP), a key component of the LPS responsible for specificity, have not been characterized in children. Here, we report a comparison of polysaccharide antibody responses in children from a region in Bangladesh where cholera is endemic, including infants (6 to 23 months, n = 15), young children (24 to 59 months, n = 14), and older children (5 to 15 years, n = 23) who received two doses of a killed oral cholera vaccine 14 days apart. We found that infants and young children receiving the vaccine did not mount an IgG, IgA, or IgM antibody response to V. cholerae OSP or LPS, whereas older children showed significant responses. In comparison to the vaccinees, young children with wild-type V. cholerae O1 Ogawa infection did mount significant antibody responses against OSP and LPS. We also demonstrated that OSP responses correlated with age in vaccinees, but not in cholera patients, reflecting the ability of even young children with wild-type cholera to develop OSP responses. These differences might contribute to the lower efficacy of protection rendered by vaccination than by wild-type disease in young children and suggest that efforts to improve lipopolysaccharide-specific responses might be critical for achieving optimal cholera vaccine efficacy in this younger age group.     

Assuming Peripheral Elimination: Its Impact on the Estimation of Pharmacokinetic Parameters of Muscle Relaxants

For anesthetic drugs undergoing nonorgan-based elimination, there is a definite trend towards using pharmacokinetic (PK) models in which elimination can occur from both central (k₁₀ ) and peripheral compartments(k₂₀ ). As the latter cannot be assessed directly, assumptions have to be made regarding its value. The primary purpose of this paper is to evaluate the impact of assuming various degrees of peripheral elimination on the estimation of PK parameters. For doing so, an explanatory model is presented where previously published data from our laboratory on three muscle relaxants, i.e., atracurium, doxacurium, and mivacurium, are used for simulations. The mathematical aspects for this explanatory model as well as for two specific applications are detailed. Our simulations show that muscle relaxants having a short elimination half-life are more affected by the presence of peripheral elimination as their distribution phase occupies the major proportion of their total area under the curve. Changes in the exit site dependent PK parameters (Vd_ss ) are also mostly significant when k₂₀ is smaller than k₁₀ . Although the physiological processes that determine drug distribution and those affecting peripheral elimination are independent, the two are mathematically tied together in the two-compartment model with both central and peripheral elimination. It follows that, as greater importance is given to k₂₀ , the rate of transfer from the central compartment (k₁₂ ) increases. However, as a result of a proportional increase in the volume of the peripheral compartment, peripheral concentrations remain unchanged whether or not peripheral elimination is assumed. These findings point out the limitations of compartmental analysis when peripheral elimination cannot be measured directly.     

Oral cholera vaccination strategy: Self-administration of the second dose in urban Dhaka,Bangladesh

Cholera remains a major public health problem in many developing countries including Bangladesh. The oral cholera vaccine (OCV) is now considered a key component of the public health response to cholera. Although maintaining cold chain and organizing human resource are the major challenges of vaccine delivery to the community. Here we applied an innovative approach to second dose OCV delivery to minimize financial and logistic burdens. The purpose of this study was to assess the feasibility and compliance of second dose self-administration when the second dose was provided in a plastic bag to first dose vaccine recipients as OCV is stable for up to 42?days at ambient temperatures. We aimed to deploy vaccines (N?=?112,000) left over from other studies to 56,000 people aged?≥?one year living in Mirpur, Dhaka to see the feasibility of self-administration strategy. During vaccination, the first OCV dose (OCV1) was given from fixed sites and the second dose (OCV2) was provided in a plastic zip-lock bag for the participant to take the vaccine two weeks later at home. Participants were instructed to keep the vaccine away from light and in a dry cool place. Empty vials were collected following the end date of the scheduled second vaccination. Of the targeted population, 41,694 (74%) received the first OCV dose whereas an estimated 38,852 (93% of those receiving the first dose) received the second dose which represents a 7% drop out rate from OCV1 to OCV2. However the average two dose coverage was 69%. A survey of a subsample 2990 (from 8551) randomly selected households revealed that almost all respondents (98.75%) appreciated this new self-administration strategy and considered the strategy to be more practical and convenient than the usual method. This simplified, self-administered delivery strategy provides an ideal alternative for second-dose OCV delivery in hard-to-reach populations and resource-poor settings.     

Assessing drug distribution in tissues expressing P-glycoprotein using physiologically based pharmacokinetic modeling: identification of important model parameters through global sensitivity analysis

The structural complexity of a PBPK model is usually accompanied with significant uncertainty in estimating its input parameters. In the last decade, the global sensitivity analysis, which accounts for the variability of all model input parameters simultaneously as well as their correlations, has gained a wide attention as a powerful probing technique to identify and control biological model uncertainties. However, the current sensitivity analysis techniques used in PBPK modeling often neglect the correlation between these input parameters. We introduce a new strategy in the PBPK modeling field to investigate how the uncertainty and variability of correlated input parameters influence the outcomes of the drug distribution process based on a model we recently developed to explain and predict drug distribution in tissues expressing P-glycoprotein (P-gp). As direct results, we will also identify the most important input parameters having the largest contribution to the variability and uncertainty of model outcomes. We combined multivariate random sampling with a ranking procedure. Monte–Carlo simulations were performed on the PBPK model with eighteen model input parameters. Log-normal distributions were assumed for these parameters according to literature and their reported correlations were also included. A multivariate sensitivity analysis was then performed to identify the input parameters with the greatest influence on model predictions. The partial rank correlation coefficients (PRCC) were calculated to establish the input–output relationships. A moderate variability of predicted C_last and C_max was observed in liver, heart and brain tissues in the presence or absence of P-gp activity. The major statistical difference in model outcomes of the predicted median values has been obtained in brain tissue. PRCC calculation confirmed the importance for a better quantitative characterisation of input parameters related to the passive diffusion and active transport of the unbound drug through the blood-tissue membrane in heart and brain. This approach has also identified as important input parameters those related to the drug metabolism for the prediction of model outcomes in liver and plasma. The proposed Monte–Carlo/PRCC approach was aimed to address the effect of input parameters correlation in a PBPK model. It allowed the identification of important input parameters that require additional attention in research for strengthening the physiological knowledge of drug distribution in mammalian tissues expressing P-gp, thereby reducing the uncertainty of model predictions.     

Complexity of rice-water stool from patients with Vibrio cholerae plays a role in the transmission of infectious diarrhea

At the International Centre for Diarrhoeal Disease Research, Bangladesh, one-half of the rice-water stool samples that were culture-positive for Vibrio cholerae did not contain motile V. cholerae by standard darkfield microscopy and were defined as darkfield-negative (DF(-)). We evaluated the host and microbial factors associated with DF status, as well as the impact of DF status on transmission. Viable counts of V. cholerae in DF(-) stools were three logs lower than in DF(+) stools, although DF(-) and DF(+) stools had similar direct counts of V. cholerae by microscopy. In DF(-) samples, non-V. cholerae bacteria outnumbered V. cholerae 10:1. Lytic V. cholerae bacteriophage were present in 90% of DF(-) samples compared with 35% of DF(+) samples, suggesting that bacteriophage may limit culture-positive patients from producing DF(+) stools. V. cholerae in DF(-) and DF(+) samples were found both planktonically and in distinct nonplanktonic populations; the distribution of organisms between these compartments did not differ appreciably between DF(-) and DF(+) stools. This biology may impact transmission because epidemiological data suggested that household contacts of a DF(+) index case were at greater risk of infection with V. cholerae. We propose a model in which V. cholerae multiply in the small intestine to produce a fluid niche that is dominated by V. cholerae. If lytic phage are present, viable counts of V. cholerae drop, stools become DF(-), other microorganisms bloom, and cholera transmission is reduced.     

Salmonella enterica Serovar Typhi-Specific Immunoglobulin A Antibody Responses in Plasma and Antibody in Lymphocyte Supernatant Specimens in Bangladeshi Patients with Suspected Typhoid Fever

Many currently available diagnostic tests for typhoid fever lack sensitivity and/or specificity, especially in areas of the world where the disease is endemic. In order to identify a diagnostic test that better correlates with typhoid fever, we evaluated immune responses to Salmonella enterica serovar Typhi (serovar Typhi) in individuals with suspected typhoid fever in Dhaka, Bangladesh. We enrolled 112 individuals with suspected typhoid fever, cultured day 0 blood for serovar Typhi organisms, and performed Widal assays on days 0, 5, and 20. We harvested peripheral blood lymphocytes and analyzed antibody levels in supernatants collected on days 0, 5, and 20 (using an antibody-in-lymphocyte-supernatant [ALS] assay), as well as in plasma on these days. We measured ALS reactivity to a serovar Typhi membrane preparation (MP), a formalin-inactivated whole-cell preparation, and serovar Typhi lipopolysaccharide. We measured responses in healthy Bangladeshi, as well as in Bangladeshi febrile patients with confirmed dengue fever or leptospirosis. We categorized suspected typhoid fever individuals into different groups (groups I to V) based on blood culture results, Widal titer, and clinical features. Responses to MP antigen in the immunoglobulin A isotype were detectable at the time of presentation in the plasma of 81% of patients. The ALS assay, however, tested positive in all patients with documented or highly suspicious typhoid, suggesting that such a response could be the basis of improved diagnostic point-of-care-assay for serovar Typhi infection. It can be important for use in epidemiological studies, as well as in difficult cases involving fevers of unknown origin.Salmonella enterica serovar Typhi (serovar Typhi) is the cause of typhoid fever, an illness that affects over 20,000,000 individuals worldwide each year, killing over 200,000 (5, 8, 16). The largest burden of typhoid fever is borne by impoverished individuals in resource-poor areas of the world. Serovar Typhi is a human-restricted invasive enteric pathogen which, after ingestion, crosses the intestinal mucosa, is taken up by gut-associated lymphoreticular tissues, and enters the systemic circulation. Both mucosal and systemic host immune responses are stimulated after infection. Serovar Typhi is an intracellular pathogen, and antibody and cell-mediated immune responses occur after infection or immunization with live oral attenuated typhoid vaccines (10, 25, 34).Diagnostic tests for typhoid fever often lack sensitivity and/or specificity, especially in areas of the world that are endemic for typhoid fever, where clinically distinguishing typhoid fever from other febrile illnesses is difficult (5, 17, 39). Microbiologic culturing of blood is approximately 30 to 70% sensitive, with the highest sensitivity being associated with an absence of prior use of antibiotics and the culturing of larger volumes of blood, features that complicate this mode of diagnosis in young children (5, 6, 8, 36). Microbiologic culturing of bone marrow aspirates is more sensitive than blood but often clinically impractical (1, 11, 12). Serum Widal assay titers are often nonspecific in endemic settings and are of limited value unless titers are markedly elevated or are analyzed for changes from acute to convalescent phases of illness (18, 33, 38). Molecular diagnostic assays including PCR are promising, but issues of practicality, contamination, and quality control have limited their use in many resource-poor areas of the world (14).Since serovar Typhi interacts with both the mucosal and the systemic immune systems, we were interested to determine whether analyses of mucosal immune responses would give improved insight into this human-restricted infection. Activated mucosal lymphocytes migrate from intestinal tissue and circulate within peripheral blood before rehoming to mucosal tissues (20, 31). This migration peaks 1 to 2 weeks after intestinal infection and may be measured by using peripheral blood mononuclear cells (PBMC) in an antibody-secreting cell (ASC) assay (19, 26) or in supernatants recovered from harvested PBMC (the “antibody in lymphocyte supernatant” [ALS] assay) (7, 31). Although ALS and ASC responses have previously been measured after immunization with oral live attenuated typhoid vaccines, detailed analyses of ALS or ASC responses in individuals with wild-type typhoid fever are lacking (21, 24). In order to gain further insight into mucosal immune responses during wild-type serovar Typhi infection, we undertook a study to characterize the serum and ALS responses to serovar Typhi among individuals with suspected typhoid fever in Bangladesh.