Measuring training effectiveness of laboratory biosafety program offered at African Center for Integrated Laboratory Training in 22 President's Emergency Plan for AIDS Relief supported countries (2008-2014).

INTRODUCTION: The African Center for Integrated Laboratory Training (ACILT) in Johannesburg, South Africa offered a laboratory biosafety program to improve laboratory biosafety practices in 22 President's Emergency Plan for AIDS Relief (PEPFAR) supported countries. This manuscript evaluates the transference of newly gained knowledge and skills to the participants' place of employment for HIV and TB diagnostic laboratory programs. It also serves as a follow-on to a previously published manuscript that measured training effectiveness for all courses offered at ACILT. METHODS: ACILT offered 20 Laboratory Biosafety and Infrastructure courses (2008-2014), also referred as biosafety course/course comprising of 14 core laboratory safety elements to 402 participants from 22 countries. In 2015, participants received 22 e-questions divided into four categories: (1) Safety Policies, (2) Management's Engagement, (3) Safety Programs and (4) Assessments of Safety Practices to determine retrospectively the training effectiveness of biosafety practices in their place of employment 6 months before and after attending their course. We used Kirkpatrick model to assess the transference of knowledge, skills and obstructive factors. RESULTS: 20% (81/402) of the participants completed the e-questionnaire. The overall percentage of positive responses indicating implementation of new safety practices increased from 50% to 84%. Improvement occurred in all four categories after attending the course, with the greatest increases in Safety Policies (67-94%) and Safety Programs (43-91%). Creating a safety committee, allocating resources, and establishing a facility safety policy were important drivers for implementing and maintaining laboratory safety practices. In addition, accredited laboratories and countries with national safety regulations or policies had a higher percentage of improvements. The most reported challenges were inadequate funding and lack of management enforcement. CONCLUSIONS: PEPFAR and other partners' investments in training institutions, such as ACILT, were effective in building sustainable country ownership to strengthen biosafety practices and were leveraged to combat zoonotic diseases and COVID-19. Although support continues at the national/regional level, a standardized, coordinated and continent-wide sustainable approach to offer a biosafety program-like ACILT is missing. Continuous offerings of biosafety programs similar to ACILT could contribute to sustainable strengthening of laboratory biosafety, QMS and pandemic preparedness.

Assessing the effect of electronic health information exchange on the completeness and validity of data for measuring viral load testing turnaround time in Nigeria.

INTRODUCTION: Implementation of health information exchange has been shown to result in several benefits which includes the improvement in the completeness and timeliness of data for public health program monitoring and surveillance. OBJECTIVE: The objective of this study was to assess the effect of implementing an electronic health information exchange (HIE) on the quality of data available to measure HIV viral load testing turnaround time (TAT) in Nigeria. METHODS: We measured viral load data validity and completeness before the implementation of electronic health information exchange, and 6 months after implementation. Records of specimens collected at 30 healthcare facilities and tested in 3 Polymerase Chain Reaction (PCR) labs were analyzed. We define data completeness as the percentage of non-missing values and measured this value by specimens and by data elements in the dataset for calculating TAT. To examine data validity, we classified TAT segments with negative values and date fields that were not in International Organization for Standardization(ISO) standard date format as invalid. Validity was measured by specimens and by each TAT segment. Pearson's chi square was used to assess for improvements in validity and completeness post implementation of HIE. RESULTS: 15,226 records of specimens were analyzed at baseline and 18,022 records of specimens analyzed at endline. Data completeness for all specimens recorded increased significantly from 47% before HIE implementation to 67% six months after implementation (p < 0.01). Data validity also increased from 90% before implementation to 91% after implementation (p < 0.01) CONCLUSION: Our study demonstrated evidence of significant improvement in the quality of data available to measure viral load turnaround time with the implementation of HIE.

Association Between Third-Trimester Tdap Immunization and Neonatal Pertussis Antibody Concentration.

Importance: Immunization with tetanus, diphtheria, and acellular pertussis (Tdap) vaccine is recommended in the United States during weeks 27 through 36 of pregnancy to prevent life-threatening infant pertussis. The optimal gestation for immunization to maximize concentrations of neonatal pertussis toxin antibodies is unknown. Objective: To determine pertussis toxin antibody concentrations in cord blood from neonates born to women immunized and unimmunized with Tdap vaccine in pregnancy and optimal gestational age for immunization to maximize concentrations of neonatal antibodies. Design, Setting, and Participants: Prospective, observational, cohort study of term neonates in Houston, Texas (December 2013-March 2014). Exposures: Tdap immunization during weeks 27 through 36 of pregnancy or no Tdap immunization. Main Outcomes and Measures: Primary outcome was geometric mean concentrations (GMCs) of pertussis toxin antibodies in cord blood of Tdap-exposed and Tdap-unexposed neonates and proportions of Tdap-exposed and Tdap-unexposed neonates with pertussis toxin antibody concentrations of 15 IU/mL or higher, 30 IU/mL or higher, and 40 IU/mL or higher, cutoffs representing quantifiable antibodies or levels that may be protective until the infant immunization series begins. Secondary outcome was the optimal gestation for immunization to achieve maximum pertussis toxin antibodies. Results: Six hundred twenty-six pregnancies (mean maternal age, 29.7 years; 41% white, 27% Hispanic, 26% black, 5% Asian, 1% other; mean gestation, 39.4 weeks) were included. Three hundred twelve women received Tdap vaccine at a mean gestation of 31.2 weeks (range, 27.3-36.4); 314 were unimmunized. GMC of neonatal cord pertussis toxin antibodies from the Tdap-exposed group was 47.3 IU/mL (95% CI, 42.1-53.2) compared with 12.9 IU/mL (95% CI, 11.7-14.3) in the Tdap-unexposed group, for a GMC ratio of 3.6 (95% CI, 3.1-4.2; P < .001). More Tdap-exposed than Tdap-unexposed neonates had pertussis toxin antibody concentrations of 15 IU/mL or higher (86% vs 37%; difference, 49% [95% CI, 42%-55%]), 30 IU/mL or higher (72% vs 17%; difference, 55% [95% CI, 49%-61%]), and 40 IU/mL or higher (59% vs 12%; difference, 47% [95% CI, 41%-54%]); P < .001 for each analysis. GMCs of pertussis toxin antibodies were highest when Tdap vaccine was administered during weeks 27 through 30 and declined thereafter, reaching a peak at week 30 (57.3 IU/mL [95% CI, 44.0-74.6]). Conclusions and Relevance: Immunization with Tdap vaccine during the third trimester of pregnancy, compared with no immunization, was associated with higher neonatal concentrations of pertussis toxin antibodies. Immunization early in the third trimester was associated with the highest concentrations.

Clinical evaluation and validation of laboratory methods for the diagnosis of Bordetella pertussis infection: Culture, polymerase chain reaction (PCR) and anti-pertussis toxin IgG serology (IgG-PT).

INTRODUCTION: The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests. METHODS: Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for ≤ 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods-pertussis culture as the "gold standard," composite reference standard analysis (CRS), and latent class analysis (LCA). RESULTS: Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough. CONCLUSIONS: Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.

Evaluation of Commercial Assays for Single-Point Diagnosis of Pertussis in the US.

BACKGROUND: Pertussis serodiagnosis is increasingly being used in the United States despite the lack of a US Food and Drug Administration-approved, commercially available assay. To better understand the utility of these assays in diagnosing pertussis, serology assays were evaluated for analytical parameters and clinical accuracy. METHODS: Forty-three antigen-antibody combinations were evaluated for single-point diagnosis of pertussis. Serum panels included sera from laboratory-confirmed cases, an international reference standard, and healthy donors. Phase I panel (n = 20) of sera was used to assess precision, linearity, and accuracy; Phase II panel (n = 226) followed with positive percent agreement (PPA) and negative percent agreement (NPA) estimates. Analytical analyses included coefficients of variation (CV) and concordance correlation coefficients (rc). RESULTS: Intra-analyst variability was found to be relatively low among samples per assay, with only 6% (78 of 1240) having CV >20%, primarily with the highly concentrated immunoglobulin (Ig)G anti-pertussis toxin (PT) specimens and IgM assays. The rc measurements to assess linearity ranged between 0.282 and 0.994, 0.332 and 0.999, and -0.056 and 0.482 for IgA, IgG, and IgM, respectively. Analytical accuracy for calibrated IgG anti-PT assays was 86%-115%. The PPA and NPA varied greatly for all assays; PPA/NPA ranges for IgA, IgG, and IgM assays, with culture and/or polymerase chain reaction positivity as control, were 29-90/13-100, 26-96/27-100, and 0-73/42-100, respectively. In IgG assays, mixing filamentous hemagglutinin antigen with PT increased PPA but decreased NPA. CONCLUSIONS: Seroassays varied substantially under both analytical and clinical parameters; however, those that were calibrated to a reference standard were highly accurate. Our findings support incorporation of calibrated pertussis seroassays to the pertussis case definition for improved diagnosis and surveillance.

Harmonization of Bordetella pertussis real-time PCR diagnostics in the United States in 2012.

Real-time PCR (rt-PCR) is an important diagnostic tool for the identification of Bordetella pertussis, Bordetella holmesii, and Bordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in the B. pertussis genome and 32 to 65 copies in B. holmesii. The CDC developed a multitarget PCR assay to differentiate B. pertussis, B. holmesii, and B. parapertussis and provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these three Bordetella species in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viable Bordetella and non-Bordetella species in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481 in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiated B. pertussis and B. holmesii and 68% and 72% identified B. parapertussis. IS481 cycle threshold (CT) values for B. pertussis samples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiated B. pertussis and B. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCR Bordetella diagnostic protocols in USPHLs compared to that of the previous survey.

Serodiagnosis as adjunct assay for pertussis infection in São Paulo, Brazil.

Pertussis remains an important public health problem in many countries despite extensive immunization. Cultures and real-time PCR (RT-PCR) assays are the recommended pertussis diagnostic tests, but they lack sensitivity at the later stage of the disease. This study introduces the IgG anti-pertussis toxin enzyme-linked immunosorbent assay (PT ELISA) in our routine diagnosis to improve disease burden estimation. Serum samples and nasopharyngeal swabs (n = 503) were collected at the same time from patients presenting with cough illness suspected of being pertussis and tested by the PT ELISA and culture and/or RT-PCR, respectively. Patients were separated into three age groups: group 1, <1 year (n = 260; mean age, 3 months), group 2, 1 to 6 years (n = 81; mean age, 3 years), and group 3, ≥7 years (n = 162; mean age, 26 years). The times (means) from cough onset to specimen collection were 16, 24, and 26 days, respectively. In group 1, 83 (82.2%) of 101 positive cases were positive for pertussis by culture/RT-PCR, while 40 (39.6%) tested positive by PT ELISA. In group 2, 6 (19.4%) of 31 positive cases were culture/RT-PCR positive, and 29 (93.6%) were seropositive. In group 3, 13 (13.8%) of 94 positive cases were positive by culture/RT-PCR and 91 (96.8%) were positive by serology. Culture/RT-PCR detected more cases of pertussis in infants (P < 0.0001), whereas the PT ELISA detected more cases in adolescents and adults (P < 0.0001). The timing between cough onset and specimen collection or recent vaccination may have partially affected our results. Serology is a suitable, cost-effective, and complementary pertussis diagnostic tool, especially among older children, adolescents, and adults during the later disease phase.

Does tetanus-diphtheria-acellular pertussis vaccination interfere with serodiagnosis of pertussis infection?

An anti-pertussis toxin (PT) IgG enzyme-linked immunosorbent assay (ELISA) was analytically validated for the diagnosis of pertussis at a cutoff of 94 ELISA units (EU)/ml. Little was known about the performance of this ELISA in the diagnosis of adults recently vaccinated with tetanus-diphtheria-acellular pertussis (Tdap) vaccine, which contains PT. The goal of this study was to determine when the assay can be used following Tdap vaccination. A cohort of 102 asymptomatic health care personnel (HCP) vaccinated with Tdap (Adacel; Sanofi Pasteur) were aged 19 to 79 years (median, 47 years) at vaccination. For each HCP, specimens were available for evaluation at 2 to 10 time points (prevaccination to 24 months postvaccination), and geometric mean concentrations (GMC) for the cohort were calculated at each time point. Among 97 HCP who responded to vaccination, a mixed-model analysis with prediction and tolerance intervals was performed to estimate the time at which serodiagnosis can be used following vaccination. The GMCs were 8, 21, and 9 EU/ml at prevaccination and 4 and 12 months postvaccination, respectively. Eight (8%) of the 102 HCP reached antibody titers of ≥94 EU/ml during their peak response, but none had these titers by 6 months postvaccination. The calculated prediction and tolerance intervals were <94 EU/ml by 45 and 75 days postvaccination, respectively. Tdap vaccination 6 months prior to testing did not confound result interpretation. This seroassay remains a valuable diagnostic tool for adult pertussis.

Comparative study of different sources of pertussis toxin (PT) as coating antigens in IgG anti-PT enzyme-linked immunosorbent assays.

In an effort to improve the reliability and reproducibility of serological assays for Bordetella pertussis, a collaborative study was conducted to compare four different sources of pertussis toxin (PT) as coating antigens in the immunoglobulin G (IgG) anti-PT enzyme-linked immunosorbent assay (ELISA). Four sources of PT were used as coating antigens in the IgG anti-PT ELISA in four different testing laboratories (labs A to D) to determine whether the different antigen preparations and different laboratories influenced assay results. A panel of 60 sera consisting of deidentified human specimens from previous vaccination trials of healthy adults and infants and clinical specimens from outbreak settings was tested. In the four laboratories, each sample was tested three times with the four PT antigens according to the standard coating optimization and IgG anti-PT ELISA testing procedures used in that laboratory. Differences among the antigens, as well as intra- and interlaboratory variability, were evaluated. Excellent agreement was observed with the test panel results among the four antigens within each laboratory. Concordance correlation coefficient (r(c)) measurements among the different antigens ranged from 0.99, 0.99 to 1.00, 1.00, and 0.97 to 1.00 for labs A to D, respectively. The comparisons between pairs of laboratories also indicated a high degree of concordance for each PT preparation, with r(c) measurements between 0.90 and 0.98, 0.93 and 0.99, 0.92 and 0.98, and 0.93 and 0.99 for antigens 1 to 4, respectively. Relatively minor differences in results were observed among laboratories or among antigens, suggesting that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine evaluation.

Effectiveness of adolescent and adult tetanus, reduced-dose diphtheria, and acellular pertussis vaccine against pertussis.

BACKGROUND: Pertussis is among the most poorly controlled bacterial vaccine-preventable diseases in the United States. In 2006, a tetanus, reduced-dose diphtheria, and acellular pertussis (Tdap) booster was recommended for adolescents and adults. Tdap vaccines were licensed on the basis of antibody response without vaccine effectiveness data. METHODS: From 30 September 2007 through 19 December 2007, a pertussis outbreak occurred at a nursery through twelfth grade school on St. Croix, US Virgin Islands. We screened all students for cough and collected clinical history, including Tdap receipt. Coughing students were offered diagnostic testing. We defined clinical case patients as students with cough 14 days in duration plus either whoop, paroxysms, or post-tussive vomiting, and we defined confirmed case patients as students with any cough with isolation of Bordetella pertussis or those with clinical cases and polymerase chain reaction or serological evidence of pertussis; other clinical cases were classified as probable. RESULTS: There were 51 confirmed or probable cases among 499 students (attack rate, 10%). Disease clustered in grades 6-12, with a peak attack rate of 38% among 10th graders. Of 266 students aged 11 years with complete data, 31 (12%) had received Tdap. Forty-one unvaccinated students (18%) had confirmed or probable pertussis, compared with 2 (6%) of the vaccinated students (relative risk, 2.9); vaccine effectiveness was 65.6% (95% confidence interval, -35.8% to 91.3%; P = .092). CONCLUSIONS: This first evaluation of Tdap vaccine effectiveness in the outbreak setting suggests that Tdap provides protection against pertussis. Increased coverage is needed to realize the full benefit of the vaccine program. Serological testing was an important tool for case identification and should be considered for inclusion in the Council of State and Territorial Epidemiologists case definition.

Development and analytical validation of an immunoassay for quantifying serum anti-pertussis toxin antibodies resulting from Bordetella pertussis infection.

Adequately sensitive and specific methods to diagnose pertussis in adolescents and adults are not widely available. Currently, no Food and Drug Administration-approved diagnostic assays are available for the serodiagnosis of Bordetella pertussis. Since concentrations of B. pertussis-specific antibodies tend to be high during the later phases of disease, a simple, rapid, easily transferable serodiagnostic test was developed. This article describes test development, initial evaluation of a prototype kit enzyme-linked immunosorbent assay (ELISA) in an interlaboratory collaborative study, and analytical validation. The data presented here demonstrate that the kit met all prespecified criteria for precision, linearity, and accuracy for samples with anti-pertussis toxin (PT) immunoglobulin G (IgG) antibody concentrations in the range of 50 to 150 ELISA units (EU)/ml, the range believed to be most relevant for serodiagnosis. The assay met the precision and linearity criteria for a wider range, namely, from 50 to 200 EU/ml; however, the accuracy criterion was not met at 200 EU/ml. When the newly adopted World Health Organization International Standard for pertussis antiserum (human) reference reagent was used to evaluate accuracy, the accuracy criteria were met from 50 to 200 international units/ml. In conclusion, the IgG anti-PT ELISA met all assay validation parameters within the range considered most relevant for serodiagnosis. This ELISA was developed and analytically validated as a user-friendly kit that can be used in both qualitative and quantitative formats. The technology for producing the kit is transferable to public health laboratories.

Argatroban for anticoagulation during cardiac surgery.

BACKGROUND: The aim of this study was to report our experience and review the published data on argatroban administration during adult cardiac surgery. METHODS: The information on all reported cases of argatroban use in adults, during cardiac surgery was reviewed, including that of the patient described here. This analysis focused on patient characteristics, type of surgery, argatroban dosing schedule, monitoring of anticoagulation and outcomes. RESULTS: Twenty-one cases have been reported. Fifteen patients underwent off-pump surgical procedures with the argatroban dose adjusted to maintain an activated clotting time (ACT) range between 200 and 300 s. Three intraoperative thrombi occurred in two patients when the ACT was <280 s. None had coagulopathy. Six cases reported the use of argatroban during on-pump cardiac surgery dosed to keep the ACT >400 s. Intraoperative thrombotic complications were not reported in this group; however, one clot in the pump was noted after the procedure when the ACT was between 300 and 350 s. All six cases required larger volumes of perioperative blood products and three had severe coagulopathy. Of the 21 cases, seven had an indication for continued anticoagulation following surgery. Four cases did not report further use of argatroban after surgery. Three patients received argatroban after surgery without complications. Recommendations for how to use argatroban during cardiac surgery are proposed. CONCLUSIONS: Argatroban, with ACT monitoring, might be safely used for anticoagulation during cardiac surgery.

Cefotetan-induced hemolytic anemia after perioperative prophylaxis.

Cephalosporin-induced hemolytic anemia is an acquired form of hemolytic anemia caused by interaction of drug with the immune system. Drug adsorption, drug-dependent antibody, and autoimmune induction are the three mechanisms of hemolysis. Cefotetan-induced hemolytic anemia (CIHA) has been described to occur through all three mechanisms. We report four cases of CIHA that occurred after appropriate perioperative use of cefotetan. All of our patients developed an acute and severe hemolytic episode that caused significant symptoms and led to hospitalization within 1-2 weeks after exposure to cefotetan. The hemolytic process was self-limited, and all our patients responded to supportive measures and blood transfusion. This report adds to our knowledge of CIHA, a rare complication of cefotetan use. Our cases suggest that cefotetan-induced acute severe hemolysis is caused by membrane modification (nonimmunologic protein adsorption) in addition to immune complex formation. Prompt diagnosis and aggressive supportive measures are essential in minimizing morbidity and mortality from hemolysis. Physicians should warn their patients about this complication. Given that hemolysis occurs when the subject is no longer under direct clinical supervision, patient awareness on how to recognize signs and symptoms of hemolysis is paramount to reducing the likelihood of this potentially lethal side effect. Finally, physicians might consider restricting cefotetan use.

Etiology of thrombocytopenia in all patients treated with heparin products.

PURPOSE: To characterize the cause of thrombocytopenia in all patients treated with heparin products, to determine the incidence of heparin-induced thrombocytopenia (HIT) in unselected hospitalized patients, and to have modern data of the magnitude of this problem. METHODS: Retrospective hospital-based cohort study. During a random 2-month period, we reviewed the medical records of all patients treated with heparin agents, screened them for thrombocytopenia, and determined the cause of it. RESULTS: Out of 674 patients who received heparin products, 110 (16%) had thrombocytopenia. The most common causes included cancer chemotherapy, surgery, sepsis, and medications. Three patients met the clinical criteria for HIT. One had antibodies for heparin-platelet factor-4, and received a direct thrombin inhibitor. The other two individuals had a clinical syndrome that resembled immune HIT, but were not tested for HIT antibodies. One suffered a thrombotic episode that led to the death of her fetus. The other died of a possible thromboembolism. CONCLUSIONS: This study provides evidence-based data for the differential diagnosis of thrombocytopenia after treatment with heparin products. Our findings suggest that increased awareness of the HIT syndrome might reduce morbidity and mortality. Patients exposed to heparin products, who develop thrombocytopenia, should not be overlooked.