Validation of immunoassays for the Chlamydia trachomatis antigen Pgp3 using a chimeric monoclonal antibody.

Seroepidemiology, or measuring antibodies to pathogens to estimate population-level exposure, can provide useful public health data. The tests used, however, often lack sufficient validation data due to absence of a gold standard. For many pathogens, serum antibodies can be detected long after resolution of infection, but infection status is often used as a gold standard for antibody positivity. To ensure that recently developed antibody tests for seroepidemiology of Chlamydia trachomatis (Ct), the causative agent of urogenital chlamydia and the blinding eye disease trachoma, have high performance, we generated a chimeric antibody to the immunodominant Ct antigen Pgp3. Two clones were selected to evaluate the test performance of three assays to measure antibodies to Pgp3: multiplex bead assay (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA). Overall, each assay demonstrated high accuracy and precision when tested using either clone, and the clones were stable when stored at - 20 °C and 4 °C for almost 2 years. The limit of detection was similar for MBA and LFA, but almost a log-fold higher (i.e. less sensitive) using ELISA. Overall, the chimeric antibodies represent stable control reagents for tests with robust performance and will facilitate deployment of these tests to other laboratories.

Evaluation of a Single Dose of Azithromycin for Trachoma in Low-Prevalence Communities.

PURPOSE: Trachoma, caused by repeated ocular infection with Chlamydia trachomatis, is the leading infectious cause of blindness worldwide and is targeted for elimination as a public health problem. We sought to determine whether a one-time azithromycin mass treatment would reduce trachomatous inflammation-follicular (TF) levels below the elimination threshold of 5% in communities with disease prevalence between 5 and 9.9%. METHODS: The study was conducted in 96 sub-village units (balozis) in the Kongwa district of Tanzania which were predicted from prior prevalence surveys to have TF between 5 and 9.9%. Balozis were randomly assigned to the intervention and control arms. The intervention arm received a single mass drug administration of azithromycin. At baseline and 12-month follow-up, ocular exams for trachoma, ocular swabs for detection of chlamydial DNA, and finger prick blood for analysis of anti-chlamydial antibody were taken. RESULTS: Comparison of baseline and 12-month follow-up showed no significant difference in the overall TF1-9 prevalence by balozi between control and treatment arms. In the treatment arm there was a significant reduction of ocular infection 12 months after treatment (p = 0.004) but no change in the control arm. No change in Pgp3-specific antibody responses were observed after treatment in the control or treatment arms. Anti-CT694 responses increased in both study arms (p = 0.009 for control arm and p = 0.04 for treatment arm). CONCLUSION: These data suggest that a single round of MDA may not be sufficient to decrease TF levels below 5% when TF1-9 is between 5 and 9.9% at baseline.

The utility of serology for elimination surveillance of trachoma.

Robust surveillance methods are needed for trachoma control and recrudescence monitoring, but existing methods have limitations. Here, we analyse data from nine trachoma-endemic populations and provide operational thresholds for interpretation of serological data in low-transmission and post-elimination settings. Analyses with sero-catalytic and antibody acquisition models provide insights into transmission history within each population. To accurately estimate sero-conversion rates (SCR) for trachoma in populations with high-seroprevalence in adults, the model accounts for secondary exposure to Chlamydia trachomatis due to urogenital infection. We estimate the population half-life of sero-reversion for anti-Pgp3 antibodies to be 26 (95% credible interval (CrI): 21-34) years. We show SCRs below 0.015 (95% confidence interval (CI): 0.0-0.049) per year correspond to a prevalence of trachomatous inflammation-follicular below 5%, the current threshold for elimination of active trachoma as a public health problem. As global trachoma prevalence declines, we may need cross-sectional serological survey data to inform programmatic decisions.

Impact of Ivermectin Mass Drug Administration for Lymphatic Filariasis on Scabies in Eight Villages in Kongwa District, Tanzania.

Scabies was recently added to the World Health Organization list of neglected tropical diseases. The ability to treat scabies with oral ivermectin makes a mass drug administration (MDA) campaign a feasible option for scabies control. Ivermectin MDA in communities endemic for lymphatic filariasis (LF) or onchocerciasis may already be having an impact on scabies. We examined the effect of ivermectin MDA for LF on scabies prevalence over 4 years in eight Tanzanian villages. At baseline, 4.4% (95% confidence interval [CI]: 3.7-5.4) of individuals tested positive for scabies, decreasing to 0.84% (95% CI: 0.51-1.4) after one round of ivermectin MDA but increased in Year 3 (2.5% [95% CI: 1.9-3.3]) and Year 4 (2.9% [95% CI: 2.2-3.8]). Most scabies cases were seen in children younger than 15 years. The data suggest that single-dose ivermectin MDA may not be effective in attaining long-term decreases when scabies prevalence is less than 5%.

Latent class modeling to compare testing platforms for detection of antibodies against the Chlamydia trachomatis antigen Pgp3.

Latent class modeling can be used to combine the results of multiple tests to compare the sensitivity and specificity of those tests in the absence of a gold standard. Seroepidemiology for chlamydia infection may be useful for determining the cumulative risk of infection within a population. Initial studies using the Chlamydia trachomatis immunodominant antigen Pgp3 have shown utility for seroepidemiology of sexually transmitted chlamydia and the eye disease trachoma. We present our latent class modeling results for comparison of antibody data obtained from three different Pgp3-based platforms - multiplex bead array, ELISA, and lateral flow assay. Sensitivity and specificity estimates from the best fitting latent class models were similar to estimates derived from those previously obtained using a nucleic acid amplification test as a gold standard for sensitivity and non-endemic pediatric specimens for specificity, although the estimates from latent class models had wider confidence intervals. The modeling process and evaluation highlighted the importance of including as many antibody tests as possible when fitting a latent class model to ensure that as many patterns as possible are available for evaluation. Future studies designed to evaluate antibody test performance in the absence of a gold standard should utilize as many tests as possible.

Comparison of Platforms for Testing Antibody Responses against the Chlamydia trachomatis Antigen Pgp3.

Antibody responses to Chlamydia trachomatis (CT) antigens may be useful tools for surveillance of trachoma by estimating cumulative prevalence of infection within a population. Data were compared from three different platforms-multiplex bead array (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA)-measuring antibody responses against the CT antigen protein plasmid gene product 3 (Pgp3). Sensitivity was defined as the proportion of specimens testing antibody positive from a set of dried blood spots from Tanzanian 1-9-year olds who were positive for CT nucleic acid of all nucleic acid amplification test (NAAT)-positive individuals (N = 103). The sensitivity of the LFA could not be determined because of the use of dried blood spots for this test; this specimen type has yet to be adapted to LFA. Specificity was defined as the proportion of sera from U.S. and Bolivian 1-9-year olds that had previously tested negative by the Chlamydia microimmunofluorescence (MIF) assay testing negative to Pgp3-specific antibodies (N = 154). The sensitivity for MBA and ELISA was the same-93.2 (95% confidence interval [CI]: 88.3-98.1). Specificity ranged across platforms from 96.1 (95% CI: 91.8-98.2) to 99.4% (95% CI: 98.2-100). ELISA performance was similar regardless of whether the plates were precoated or freshly coated with antigen. Sensitivity and specificity of control panels were similar if the cutoff was determined using receiver operator curves or a finite mixture model, but the cutoffs themselves differed by approximately 0.5 OD using the different methodologies. These platforms show good sensitivity and specificity and show good agreement between tests at a population level, but indicate variability for ELISA outcomes depending on the cutoff determination methodology.

Evaluation of Multiplex-Based Antibody Testing for Use in Large-Scale Surveillance for Yaws: a Comparative Study.

WHO has targeted yaws for global eradication by 2020. The program goals are to interrupt the transmission in countries where yaws is endemic and to certify countries as yaws free where yaws was endemic in the past. No new rapid plasmin reagin (RPR) seroreactivity in young children is required for certification of elimination at a country level. We sought to evaluate whether antibody responses to specific treponemal antigens measured in a high-throughput multiplex bead array (MBA) assay differentiate past versus current infection and whether a nontreponemal lipoidal antigen test can be incorporated into the MBA. Serum and dried blood spot specimens collected for yaws surveillance projects in Ghana, Vanuatu, and Papua New Guinea (PNG) were run on MBA to measure antibodies against recombinant p17 (rp17) and treponemal membrane protein A (TmpA) treponemal antigens. Results were compared to standard treponemal laboratory (TPPA or TPHA [TPP(H)A]) and quantitative RPR test data. Of 589 specimens, 241 were TPP(H)A(+)/RPR(+), 88 were TPP(H)A(+)/RPR(-), 6 were TPP(H)A(-)/RPR(+), and 254 were negative for both tests. Compared to TPP(H)A, reactive concordance of rp17 was 93.7%, while reactive concordance of TmpA was only 81.9%. TmpA-specific reactivity showed good correlation with RPR titers (R(2) = 0.41; P < 0.0001). IgG responses to the lipoidal antigen used in RPR testing (cardiolipin) were not detected in the MBA. Our results suggest that TmpA can be used as a treponemal antigen marker for recent or active infection and potentially replace RPR in a high-throughput multiplex tool for large-scale yaws surveillance.

Serological Measures of Trachoma Transmission Intensity.

Ocular infection with Chlamydia trachomatis can lead to trachoma, a leading infectious cause of blindness. Trachoma is targeted for elimination by 2020. Clinical grading for ocular disease is currently used for evaluating trachoma elimination programs, but serological surveillance can be a sensitive measure of disease transmission and provide a more objective testing strategy than clinical grading. We calculated the basic reproduction number from serological data in settings with high, medium, and low disease transmission based on clinical disease. The data showed a striking relationship between age seroprevalence and clinical data, demonstrating the proof-of-principle that age seroprevalence predicts transmission rates and therefore could be used as an indicator of decreased transmission of ocular trachoma.

Trypanosoma cruzi survival following cold storage: possible implications for tissue banking.

While Trypanosoma cruzi, the etiologic agent of Chagas disease, is typically vector-borne, infection can also occur through solid organ transplantation or transfusion of contaminated blood products. The ability of infected human cells, tissues, and cellular and tissue-based products (HCT/Ps) to transmit T. cruzi is dependent upon T. cruzi surviving the processing and storage conditions to which HCT/Ps are subjected. In the studies reported here, T. cruzi trypomastigotes remained infective 24 hours after being spiked into blood and stored at room temperature (N = 20); in 2 of 13 parasite-infected cultures stored 28 days at 4°C; and in samples stored 365 days at -80°C without cryoprotectant (N = 28), despite decreased viability compared to cryopreserved parasites. Detection of viable parasites after multiple freeze/thaws depended upon the duration of frozen storage. The ability of T. cruzi to survive long periods of storage at +4 and -80°C suggests that T. cruzi-infected tissues stored under these conditions are potentially infectious.

Regional variation in the correlation of antibody and T-cell responses to Trypanosoma cruzi.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a major cause of morbidity and mortality in Central and South America. Geographic variations in the sensitivity of serologic diagnostic assays to T. cruzi may reflect differences in T. cruzi exposure. We measured parasite-specific T-cell responses among seropositive individuals in two populations from South America with widely varying antibody titers against T. cruzi. Antibody titers among seropositive individuals were significantly lower in Arequipa, Peru compared with Santa Cruz, Bolivia. Similarly, the proportion of seropositive individuals with positive T-cell responses was lower in Peru than Bolivia, resulting in overall lower frequencies of interferon-γ (IFNγ)-secreting cells from Peruvian samples. However, the magnitude of the IFNγ response was similar among the IFNγ responders in both locations. These data indicate that immunological discrepancies based on geographic region are reflected in T-cell responses as well as antibody responses.

Clinical and immunological aspects of post-kala-azar dermal leishmaniasis in Bangladesh.

We conducted active surveillance for kala-azar and post-kala-azar dermal leishmaniasis (PKDL) in a population of 24,814 individuals. Between 2002 and 2010, 1,002 kala-azar and 185 PKDL cases occurred. Median PKDL patient age was 12 years; 9% had no antecedent kala-azar. Cases per 10,000 person-years peaked at 90 for kala-azar (2005) and 28 for PKDL (2007). Cumulative PKDL incidence among kala-azar patients was 17% by 5 years. Kala-azar patients younger than 15 years were more likely than older patients to develop PKDL; no other risk factors were identified. The most common lesions were hypopigmented macules. Of 98 untreated PKDL patients, 48 (49%) patients had resolution, with median time of 19 months. Kala-azar patients showed elevated interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), and interleukin 10 (IL-10). Matrix metalloproteinase 9 (MMP9) and MMP9/tissue inhibitor of matrix metalloproteinase-1 (TIMP1) ratio were significantly higher in PKDL patients than in other groups. PKDL is frequent in Bangladesh and poses a challenge to the current visceral leishmaniasis elimination initiative in the Indian subcontinent.