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ABSTRACT Serological assays detecting IgM antibodies in addition to IgG antibodies have a diagnostic advantage in finding early infections. Staphylococcal protein A (SpA), widely used as an antibody-detecting reagent in various immunoassays, is considered to have a high binding affinity mainly to IgG, although its interaction with other classes of immunoglobulins has also been documented. Using 28 samples from 22 HIV-1 seroconversion panels, the present study demonstrated detection of early IgM antibodies by SpA-based rapid point-of-care tests, including DPP HIV 1/2, DPP HIV-Syphilis, STAT-PAK HIV 1/2, and Sure Check HIV 1/2. Samples with predominant IgM antibodies were identified by in-house IgM assays and confirmed by pretreatment with 0.1 M 2-mercaptoethanol. Likewise, the detection of treponemal IgM antibodies was shown by DPP HIV-Syphilis assay in eight samples collected at early syphilis infection. Direct interaction between IgM and SpA immobilized in solid phase or in solution was demonstrated with purified human polyclonal IgM. A strong correlation was found between the antibody levels detected by SpA and anti-IgM reagent in the early seroconversion samples, thus supporting the evidence for IgM binding by SpA. These assays demonstrated the ability to detect IgM antibodies, which may increase test sensitivity in early infections due to a reduced serodiagnostic window. IMPORTANCE Sexually transmitted infections, including HIV and syphilis, remain a global public health concern. The main laboratory testing approach for HIV and syphilis relies on serological assays. Detection of the IgM class of antibodies may have a diagnostic advantage in finding early infections. The present study using well-characterized HIV-1 and syphilis samples has demonstrated that staphylococcal protein A employed for antibody detection in rapid point-of-care tests, including DPP HIV 1/2, DPP HIV-Syphilis, STAT-PAK HIV 1/2, and Sure Check HIV 1/2, can capture IgM antibodies in addition to IgG antibodies. The findings strongly suggest that the ability to detect IgM antibodies by these immunoassays may facilitate the identification of acute-stage HIV and syphilis infections.

ABSTRACT There is a critical need for an improved rapid diagnostic for enteric fever. We have previously demonstrated that serum IgA responses targeting Salmonella enterica serovar Typhi hemolysin E (HlyE) and lipopolysaccharide (LPS) are able to discriminate patients with acute typhoid from healthy controls in areas where enteric fever is endemic (healthy endemic controls) and from patients with other bacterial infections. We now have data demonstrating that IgA antibody responses against these antigens also work well for identifying patients with acute S. Paratyphi A infection. To develop a test for acute enteric fever detection, we have adapted a point-of-care immunochromatographic dual-path platform technology (DPP), which improves on the traditional lateral flow technology by using separate sample and conjugate paths and a compact, portable reader, resulting in diagnostics with higher sensitivity and multiplexing abilities. In this analysis, we have compared our standard enzyme-linked immunosorbent assay (ELISA) method to the DPP method in detecting acute phase plasma/serum anti-HlyE and anti-LPS IgA antibodies in a cohort of patients with culture-confirmed S. Typhi (n = 30) and Paratyphi A infection (n = 20), healthy endemic controls (n = 25), and febrile endemic controls (n = 25). We found that the DPP measurements highly correlated with ELISA results, and both antigens had an area under the curve (AUC) of 0.98 (sensitivity of 92%, specificity of 94%) with all controls and an AUC of 0.98 (sensitivity of 90%, specificity of 96%) with febrile endemic controls. Our results suggest that the point-of-care DPP Typhoid System has high diagnostic accuracy for the rapid detection of enteric fever and warrants further evaluation. IMPORTANCE Enteric fever remains a significant global problem, and control programs are significantly limited by the lack of an optimal assay for identifying individuals with acute infection. This is especially critical considering the recently released World Health Organization (WHO) position paper endorsing the role of the typhoid conjugate vaccine in communities where enteric fever is endemic. A reliable diagnostic test is needed to assess and evaluate typhoid intervention strategies and determine which high-burden areas may benefit most from a vaccine intervention. Our collaborative team has developed and evaluated a point-of-care serodiagnostic assay based on detection of anti-HlyE and LPS IgA. Our finding of the high diagnostic accuracy of the DPP Typhoid System for the rapid detection of enteric fever has the potential to have significant public health impact by allowing for improved surveillance and for control and prevention programs in areas with limited laboratory capacity.

Abstract Background Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. Results Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP®) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. Conclusions Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds.

Background: To eradicate yaws, national control programmes use the Morges strategy (initial mass treatment and biannual resurveys). The resurvey component is designed to actively detect and treat remaining yaws cases and is initiated on the basis of laboratory-supported reactive non-treponemal serology (using the rapid plasma reagin [RPR] test). Unfortunately, the RPR test is available rarely in yaws-endemic areas. We sought to assess a new point-of-care assay—the Dual Path Platform (DPP) syphilis assay, which is based on simultaneous detection of antibodies to treponemal and non-treponemal antigens—for guiding use of antibiotics for yaws eradication. A secondary goal was to ascertain at what timepoint the DPP assay line reverted to negative after treatment. Methods: 703 children (aged 1–18 years) with suspected clinical yaws living in two remote, yaws-endemic villages in Papua New Guinea were enrolled. Clinical suspicion of yaws was established according to a WHO pictorial guide. We obtained blood samples from all patients. We calculated the sensitivity and specificity of the DPP assay for detection of antibodies to treponemal (T1) and non-treponemal (T2) antigens and compared values against those obtained with standard laboratory tests (the Treponema pallidum haemagglutination assay [TPHA] and the RPR test). We followed up a subsample of children with dually positive serology (T1 and T2) to monitor changes in DPP optical density (using an automatic reader) at 3 and 6 months. This trial is registered with ClinicalTrials.gov, number NCT01841203. Findings: Of 703 participants, 389 (55%) were reactive for TPHA, 305 (43%) for the RPR test, and 287 (41%) for both TPHA and the RPR test. The DPP T1 (treponemal) assay had a sensitivity of 88·4% (95% CI 84·8–91·4) and specificity of 95·2% (92·2–97·3). The DPP T2 (non-treponemal) assay had a sensitivity of 87·9% (83·7–91·3) and specificity of 92·5% (89·4–94·9). In subgroup analyses, sensitivities and specificities did not differ according to type of specimen (plasma vs whole blood). For specimens with an RPR titre of 1:8 or greater, the sensitivity of the DPP T2 assay was 94·1% (95% CI 89·9–96·9). Serological cure (including seroreversion or a fourfold reduction in optical density value) was attained at 6 months in 173 (95%) of 182 children with dual-positive serology. Interpretation: The DPP assay is accurate for identification of antibodies to treponemal and non-treponemal antigens in patients with yaws and avoids the need for laboratory support. A change of diagnostic procedure from the currently implemented RPR test to the simpler DPP assay could ease the implementation of yaws eradication activities. Funding: Chembio Diagnostic Systems, Newcrest Mining, and the Papua New Guinea National Department of Health.

BACKGROUND:Diagnosis of leptospirosis by the gold standard serologic assay, the microscopic agglutination test (MAT), requires paired sera and is not widely available. We developed a rapid assay using immunodominant Leptospira immunoglobulin-like (Lig) proteins in a Dual Path Platform (DPP). This study aimed to evaluate the assay's diagnostic performance in the setting of urban transmission. METHODOLOGY:We determined test sensitivity using 446 acute and convalescent sera from MAT-confirmed case-patients with severe or mild leptospirosis in Brazil. We assessed test specificity using 677 sera from the following groups: healthy residents of a Brazilian slum with endemic transmission, febrile outpatients from the same slum, healthy blood donors, and patients with dengue, hepatitis A, and syphilis. Three operators independently interpreted visual results without knowing specimen status. RESULTS:The overall sensitivity for paired sera was 100% and 73% for severe and mild disease, respectively. In the acute phase, the assay achieved a sensitivity of 85% and 64% for severe and mild leptospirosis, respectively. Within seven days of illness onset, the assay achieved a sensitivity of 77% for severe disease and 60% for mild leptospirosis. Sensitivity of the DPP assay was similar to that for IgM-ELISA and increased with both duration of symptoms (chi-square regression P = 0.002) and agglutinating titer (Spearman ρ = 0.24, P

A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of mycobacteriosis. In the lungs, multiple tuberculoid granulomas communicating with the bronchiolar lumen, pleural effusion, and a granulomatous lymphadenitis involving mediastinal and tracheobronchial lymph nodes were found. Serologic response to M. tuberculosis antigens was detected in the infected horse, but not in the group of 42 potentially exposed animals (18 horses, 14 alpacas, 6 donkeys, and 4 dogs) which showed no signs of disease. Diagnosis of tuberculosis in live horses remains extremely difficult. Four of 20 animal handlers at the farm were positive for tuberculous infection upon follow-up testing by interferon-gamma release assay, indicating a possibility of interspecies transmission of M. tuberculosis.

In 2009, Mycobacterium bovis infection was detected in a herd of 60 elk (Cervus elaphus) and 50 fallow deer (Dama dama) in Nebraska, USA. Upon depopulation of the herd, the prevalence of bovine tuberculosis (TB) was estimated at ∼71–75%, based upon histopathology and culture results. Particularly with elk, gross lesions were often severe and extensive. One year ago, the majority of the elk had been tested for TB by single cervical test (SCT), and all were negative. After initial detection of a tuberculous elk in this herd, 42 of the 59 elk were tested by SCT. Of the 42 SCT-tested elk, 28 were TB-infected with only 3/28 reacting upon SCT. After SCT, serum samples were collected from the infected elk and fallow deer from this herd at necropsy and tested by three antibody detection methods including multiantigen print immunoassay, cervidTB STAT-PAK, and dual path platform VetTB (DPP). Serologic test sensitivity ranged from 79 to 97% depending on the test format and host species. Together, these findings demonstrate the opportunities for use of serodiagnosis in the rapid detection of TB in elk and fallow deer.

BACKGROUND: Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75-85%, specificity of 70-92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms. METHODOLOGY/PRINCIPAL FINDINGS: Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83-99.61%; K39: 84.30-98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53-98.95%; K39: 85.54-96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46-99.1 in Sudan and 98.1% [95% CI = 89.93-99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25-93.23%] in Sudan and 88.7% [95% CI = 76.97-95.73%] in Bangladesh). CONCLUSIONS/SIGNIFICANCE: Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care.