Novel paradigm enables accurate monthly gestational screening to prevent congenital toxoplasmosis and more

Abstract

Background: Congenital toxoplasmosis is a treatable, preventable disease, but untreated causes death, prematurity, loss of sight, cognition and motor function, and substantial costs worldwide.

Objectives: We asked whether high performance of an Immunochromatographic-test (ICT) could enable accurate, rapid diagnosis/treatment, establishing new, improved care-paradigms at point-of-care and clinical laboratory.

Methods: Data were obtained in 12 studies/analyses addressing: 1-feasibility/efficacy; 2-false-positives; 3-acceptability; 4-pink/black-line/all studies; 5-time/cost; 6-Quick-Information/Limit-of-detection; 7, 8-acute;-chronic; 9-epidemiology; 10-ADBio; 11,12-Commentary/Cases/Chronology.

Findings: ICT was compared with gold-standard or predicate-tests. Overall, ICT performance for 1093 blood/4967 sera was 99.2%/97.5% sensitive and 99.0%/99.7% specific. However, in clinical trial, FDA-cleared-predicate tests initially caused practical, costly problems due to false-positive-IgM results. For 58 persons, 3/43 seronegative and 2/15 chronically infected persons had false positive IgM predicate tests. This caused substantial anxiety, concerns, and required costly, delayed confirmation in reference centers. Absence of false positive ICT results contributes to solutions: Lyon and Paris France and USA Reference laboratories frequently receive sera with erroneously positive local laboratory IgM results impeding patient care. Therefore, thirty-two such sera referred to Lyon's Reference laboratory were ICT-tested. We collated these with other earlier/ongoing results: 132 of 137 USA or French persons had false-positive local laboratory IgM results identified correctly as negative by ICT. Five false positive ICT results in Tunisia and Marseille, France, emphasize need to confirm positive ICT results with Sabin-Feldman-Dye-test or western blot. Separate studies demonstrated high performance in detecting acute infections, meeting FDA, CLIA, WHO REASSURED, CEMark criteria and patient and physician satisfaction with monthly-gestational-ICT-screening.

Conclusions/significance: This novel paradigm using ICT identifies likely false positives or raises suspicion that a result is truly positive, rapidly needing prompt follow up and treatment. Thus, ICT enables well-accepted gestational screening programs that facilitate rapid treatment saving lives, sight, cognition and motor function. This reduces anxiety, delays, work, and cost at point-of-care and clinical laboratories.

Trial registration: NCT04474132, https://clinicaltrials.gov/study/NCT04474132 ClinicalTrials.gov.

Fig 1. Design, “Roadmap” and Context for Study 1 Performed herein.

Photograph of hands and kit from [23] open access license. Map produced in Carta. From [8]. Open Access License.

Fig 2. High performance of ICT. A. Clinical Feasibility Trial.

Solid symbols represent positive predicate test, open circles represent negative predicate test. Small black dot indicates false positive predicate test. Solid colored symbols indicate a positive test. Open symbols indicate negative test result. Each of the three testers is indicated by a different color in this figure* False positive IgM predicate test for seropositive person. B. ICT is negative with 32 false positive standard tests in Lyon. C. ICT is negative with false positive standard tests in Paris, Blue IgG, green IgM. D. ADBIO, a USA test, suffers from false negative and positive IgM results. E. ICT detects acute infections with positive IgG and IgM in Colombia. F LOD Quick Instructions and Limit of Detection Study 6 Results. Box 3 contains Design and detail of methods and results. Quick instructions are able to teach 9 testers, 3 physicians, 3 medical students and 3 nurses to use test accurately with 7 samples that are negative and 7 samples at the limits of detection. Details of each study are also in Tables and Figures as follows in parentheses: Fig 2A (Study 1, Fig 1A–1E, Table 1 and A in S1 Commentary); B. (Study 2a, Table 1 and Table B in S1 Commentary); C. (Study 2b and Box 2); D. (Study 10, Table 5); E. (Study 7, Table 5); F. Box 3 and its legend provide detail for Fig 2F (Study 6). Limits of Detection and Quick Information, Box 3-Part A is the actual Quick Information (QI) provided, Photograph of hand and kit from [23] open access license. Box 3 Part B shows the study design, Box 3 Part C shows examples of test results for two testers, Box 3D shows the tabulated results of all the readers in real time and of the photographs). Box 3 Legend provides Study 6’s. structure and results. In this study we demonstrate the ease in learning to use a simple, carefully designed test description and that these specific instructions which were prepared in accordance with FDA and CLIA requirements and review (A), can function as an effective teaching tool (B, C). This study 6 is also pertinent to demonstrating repeatability and reproducibility of this test. In (B), whole blood was tested at the defined limits of detection of the sample, with 14 negative or positive samples. Positive or negative status is blinded for these 9 independent tester/readers plus additional blinded reader. For this study, these 9 testers were without other training with the ICT and worked in 3 different locations. (B, C). Performance was perfect for each of them after reading this page of instructions (B, C). This is consonant with extensive and rigorous testing of use of this ICT in a variety of USA settings, as well as in other countries in many settings with minimal instruction. Detail of objective, methods, data content of Study 6 also is in the accompanying Box. As outlined in the Box, Study 6 shown in 2F, demonstrates that it is easy for physicians, physicians in training, and nurses to learn to use a simple carefully designed description (Quick Information, QI) shown in (A). The QI is about use of the ICT. This study and its results demonstrate that this QI is an effective teaching tool. Also, we find that results with the ICT are robust, repeatable, reproducible, sensitive and specific in multiple settings. Box 2 and Tables 1, 2 and 3 and Box 3 and Perspectives section of Discussion, and Supplement (S1 Commentary) contain additional pertinent information and data.

Fig 3

A. Graphic showing monthly test method. Use of ICT in NRL as considered originally in and modified from our earlier work (33). Help refers to seeking expert guidance and reference laboratory assistance. B. Role of ICT in this algorithm shown diagrammatically. C. Representative example of ICT detecting very early seroconversion using sera originally stored for another purpose tested retrospectively, showing contrast with false positives. First pregnancy by IVF. The testing was all done retrospectively after positive IgG, and IgM at ~12–14 weeks gestation was discovered, At this time the ICT was an experimental research laboratory test done prior to studies performed for consideration of FDA clearance, CLIA waiver process and not for clinical care. However, they demonstrated, in this unusual circumstance, sensitivity early in this true seroconversion. This was while the Sabin Feldman Dye test that detects IgG directed against T.gondii and other Palo Alto Specialty laboratory tests including the IgM ELISAs were negative (antedating the time that they later became positive documenting seroconversion). Details with dates were as follows: December 2018. Began planning for IVF-sera Toxoplasma gondii antibody negative (Neg). March 2019. IVF embryos harvested cryopreserved-sera Toxoplasma gondii antibody negative. March 2019. Patient traveled to New Zealand returned end of March to USA. April 6- Patient sera obtained pre-transfer of embryos. Palo Alto reported negative g and m but there was for the first-time background in IgM ELISA, (~ 0.2, cut off ~2 for an adult), ENZO lab at NYU hospital -commercial lab reported T.gondii specific IgM positive. Since the commercial IgM was not considered reliable because of known false positives, the serum sample was sent to Palo Alto where this was found to be negative(neg). April 8 -Serum samples sent to University of Chicago Research laboratory, were tested retrospectively at a later time. ICT was weakly, but clearly positive *; Note this was pre-implantation of the embryo and IVF was in March before infection. This test is binary and not quantitative. April 9- Embryo transferred to woman. April 16-Sera hormones showed embryo implanted. ICT from this time was weakly positive when tested later, and was a little stronger than in the first sample. Five observers confirmed these readings. This was all with the same lot of the ICT. April 22- ICT a little stronger positive. Dye test 1.64, M~9, Avidity low, AC/HS acute. Subsequent sera also positive with significant rise in dye test titer and acute patterns for other tests. Pregnant woman began spiramycin at ~12 weeks gestation when the first IgG dye test, and IgM ELISA were positive. Other sera collected and tested retrospectively. Amniocentesis at 18 weeks, tested at Remington Specialty Laboratory. PCR was negative. All obstetrical ultrasounds were normal. Infant was uninfected. Abbreviations: neg is negative, + is positive, nd is not done. SFDT is Sabin Feldman Dye Test. IVF, in vitro fertilization. “wild screening” is a colloquial term referring to non-systematic testing.

Box2_Figure

Box3_Figure

Fig 4. Feasibility and acceptability of monthly screening in U.S. Academic obstetric practice.

Abbreviations in A. “neg” indicates negative results in tests. Note congruence of ICT and Reference Laboratory test conclusions. ^* Elective termination secondary to fetal anomalies, ^** Spontaneous abortion, ^*** Elected to leave the study after first test time due to traditional beliefs regarding having blood drawn. “md” indicates a missed appointment, as patient did not attend her regularly scheduled appointment. Came at alternative time and we did not connect for testing and/or survey. “n/a” indicates not available. “L&D” indicates that the patient’s monthly test was missed due to concern for premature labor, which resulted in a visit to the emergency department and then labor and delivery. “E” indicates an equivocal result, in which a barely visible band appeared which was not reproducible upon photographing the test. Per manufacturer instructions, this test was interpreted as negative. +Survey sent later, not included on graph as was not at initially planned time of six-week postpartum visit^a. A. Times and results of monthly screening for each participant. B. Survey questions and Likert scale. n the survey, number responses were considered as follows: strongly agree was 5, 4 was agree, 3 somewhat agree, 2 was somewhat disagree, 1 was strongly disagree C. Results of satisfaction survey for 14 participants. Each respondent’s answer is represented by the circles in the figure. The mean is indicated by the horizontal line, with error bars indicating standard deviation. In response to questions 1 and 4, 13/14 respondents indicated that they “strongly agree” that they would pursue testing for T. gondii in future pregnancies with POC testing, and that knowledge of T. gondii is important for pregnant women. Results were more mixed if testing required venipuncture, as indicated by the responses to questions 2 and 3, but most agreed that the test was important, would have it again in a subsequent pregnancy, and would recommend this to family and friends. It was noteworthy that other family members such as fathers of the fetus asked to be tested, relevant to the possibility of retinal disease. Right panel showed results after the time of the 6 week post-partum visit. `We did not conduct a formal survey at the time; however, we began with two providers, and other providers in the practice asked to join with their patients. Providers continued in the further analysis of the ICT as it moved toward clearance and waiver.

Fig 5. Location of seropositive persons in Cincinnati and associated demographic factors such as socioeconomic status, maximum educational level achieved, pet ownership, and ethnicity.

Sera were collected between 2017 and 2019. The low prevalence of seropositivity did not allow testing for clustering by any known risk factors for infection, including proximity to watersheds associated with sewage water run-off. None of the sociodemographic parameters, neighborhood deprivation, nor residential latitude and longitude measures achieved statistical significance. The measures of neighborhood deprivation are indicated by the color of the symbols. This map is generated from OpenStreetMap(https://www.openstreetmap.org/search?query=cincinnati#map=12/39.1506/-84.5007). OpenStreetMap data is available under the Open Database License, compatible with CC BY 4.0 license.

Fig 6. Summary of purpose and results of each study in a Roadmap of the Studies.

Flow diagram of studies that provide roadmap to step changes leading to paradigm shift in prevention of congenital toxoplasmosis.