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

Background: Congenital toxoplasmosis is a treatable, preventable disease, but untreated causes death, prematurity, loss of sight, cognition and motor function, and substantial costs worldwide. Methods/Findings: In our ongoing USA feasibility/efficacy clinical trial, data collated with other ongoing and earlier published results proved high performance of an Immunochromatographic-test(ICT) that enables accurate, rapid diagnosis/treatment, establishing new paradigms for care. Overall results from patient blood and/or serum samples tested with ICT compared with gold-standard-predicate-test results found ICT performance for 4606 sera/1876 blood, 99.3%/97.5% sensitive and 98.9%/99.7% specific. However, in the clinical trial the FDA-cleared-predicate test 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 ASSURED, 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. Author's Summary: Toxoplasmosis is a major health burden for developed and developing countries, causing damage to eyes and brain, loss of life and substantial societal costs. Prompt diagnosis in gestational screening programs enables treatment, thereby relieving suffering, and leading to > 14-fold cost savings for care. Herein, we demonstrate that using an ICT that meets WHO ASSURED-criteria identifying persons with/without antibody to Toxoplasma gondii in sera and whole blood with high sensitivity and specificity, is feasible to use in USA clinical practice. We find this new approach can help to obviate the problem of detection of false positive anti- T.gondii IgM results for those without IgG antibodies to T.gondii when this occurs in present, standard of care, predicate USA FDA cleared available assays. Thus, this accurate test facilitates gestational screening programs and a global initiative to diagnose and thereby prevent and treat T.gondii infection. This minimizes likelihood of false positives (IgG and/or IgM) while maintaining maximum sensitivity. When isolated IgM antibodies are detected, it is necessary to confirm and when indicated continue follow up testing in ∼2 weeks to establish seroconversion. Presence of a positive ICT makes it likely that IgM is truly positive and a negative ICT makes it likely that IgM will be a false positive without infection. These results create a new, enthusiastically-accepted, precise paradigm for rapid diagnosis and validation of results with a second-line test. This helps eliminate alarm and anxiety about false-positive results, while expediting needed treatment for true positive results and providing back up distinguishing false positive tests.

Clinical predictors of donor antibody titre and correlation with recipient antibody response in a COVID-19 convalescent plasma clinical trial.

BACKGROUND: Convalescent plasma therapy for COVID-19 relies on transfer of anti-viral antibody from donors to recipients via plasma transfusion. The relationship between clinical characteristics and antibody response to COVID-19 is not well defined. We investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. METHODS: Multivariable analysis of clinical and serological parameters in 103 confirmed COVID-19 convalescent plasma donors 28 days or more following symptom resolution was performed. Mixed-effects regression models with piecewise linear trends were used to characterize serial antibody responses in 10 convalescent plasma recipients with severe COVID-19. RESULTS: Donor antibody titres ranged from 0 to 1 : 3892 (anti-receptor binding domain (RBD)) and 0 to 1 : 3289 (anti-spike). Higher anti-RBD and anti-spike titres were associated with increased age, hospitalization for COVID-19, fever and absence of myalgia (all P < 0.05). Fatigue was significantly associated with anti-RBD (P = 0.03). In pairwise comparison amongst ABO blood types, AB donors had higher anti-RBD and anti-spike than O donors (P < 0.05). No toxicity was associated with plasma transfusion. Non-ECMO recipient anti-RBD antibody titre increased on average 31% per day during the first three days post-transfusion (P = 0.01) and anti-spike antibody titre by 40.3% (P = 0.02). CONCLUSION: Advanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titre to COVID-19. Despite variability in donor titre, 80% of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. A more complete understanding of the dose-response effect of plasma transfusion amongst COVID-19-infected patients is needed.

Rapid identification of positive blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry using prewarmed agar plates.

This study describes an inexpensive and straightforward method for identifying bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) directly from positive blood cultures using prewarmed agar plates. Different inoculation methods and incubation times were evaluated to determine the optimal conditions. The two methods using pelleted material from positive culture bottles performed best. In particular, the pellet streak method correctly identified 94% of the Gram negatives following 4 h of incubation and 98% of the Gram positives following 6 h of incubation.

Evaluation of FilmArray and Verigene systems for rapid identification of positive blood cultures.

The Verigene tests for Gram-positive and Gram-negative organisms in blood culture and the FilmArray blood culture identification panel were assessed for their ability to identify pathogens from positive blood cultures. Both platforms correctly identified bacteria in 92% of monomicrobial cultures analyzed, with times to identification that were significantly shorter than those for identification from subcultures.

Laboratory diagnosis of mycobacterial infections.

This paper reviews the recent US history of infection with Mycobacterium tuberculosis and discusses the emergence of drug-resistant strains. The paper continues with brief discussions of the clinical presentation of tuberculosis, tuberculosis in the pediatric population, and nontuberculous pulmonary disease. We discuss laboratory techniques that will rapidly identify Mycobacterium tuberculosis and provide susceptibility test results. The advances and limitations of currently available diagnostic techniques are presented. The chapter concludes with an explanation of the current strengths and limitations of molecular diagnosis of disease and resistance.

Sustained bacteremia associated with transjugular intrahepatic portosystemic shunt (TIPS).

Transjugular intrahepatic portosystemic shunt (TIPS) has become a routine procedure in patients with portal hypertension, yet there are few data concerning the incidence of bacteremia associated with this shunt. All patients who underwent TIPS placement at a university hospital from January 1992 through January 1999 were studied. Ninety-nine TIPS were placed, and 10 patients subsequently developed sustained bacteremia; 5 patients had no identifiable source of bacteremia despite rigorous evaluation and were presumed to represent TIPS infections, for an estimated annual incidence of 7 cases/1000 TIPS procedures. Case patients developed bacteremia a median of 100 days after TIPS placement (range, 6-732 days). Bacteremia resolved in all patients after treatment with appropriate intravenous antibiotics (median, 2 weeks of therapy). Although the incidence of TIPS-associated bacteremia appears low, the increasing frequency of this procedure suggests that more information is needed to define this entity and to develop appropriate treatment recommendations.

Yersinia enterocolitica and Yersinia pseudotuberculosis.

Yersinia enterocolitica can cause enteritis, right lower-quadrant pain mimicking appendicitis, reactive arthritis, and erythema nodosum. This organism is transmitted through food, animal contact, and contaminated blood products. Patients with iron excess are at a higher risk for serious infection. This article describes the history, microbiology, virulence factors, epidemiology, clinical manifestations, diagnosis, and therapy of Y. enterocolitica and Y. pseudotuberculosis. In addition, the immune response of those developing reactive arthritis following infection with Y. enterocolitica is discussed.

Mycobacterial growth and bacterial contamination in the mycobacteria growth indicator tube and BACTEC 460 culture systems.

The BACTEC 460 system currently provides the most rapid detection of mycobacterial growth, but the system is radiometric and requires needles to inoculate specimens through the bottle's septum. The Mycobacteria Growth Indicator Tube (MGIT) system has a liquid medium, like the BACTEC system, and does not require needles when inoculating specimens. We compared mycobacterial growth from 510 specimens in the two systems. Average time to acid-fast bacillus (AFB) detection and identification to the species level was less with the BACTEC system, but this result was statistically significant only for AFB detection in specimens containing Mycobacterium avium-M. intracellulare complex. The contamination rate with MGIT was 29%; the BACTEC rate was 5%. To investigate MGIT contamination, we initiated a second study with changes in specimen processing. The MGIT contamination rate was reduced to 12%; the BACTEC rate was not significantly affected (5.5%). The most likely explanation for the contamination in MGIT is the richness of its medium compared to the BACTEC medium. Cost analysis for the two systems in a laboratory that processes 4,500 specimens a year is presented. The data suggest that the BACTEC 460 and the MGIT systems are approximately equivalent in cost and ability to support the growth of AFB. The MGIT system appears safer and easier to use and was preferred by laboratory personnel, but it cannot currently be used for blood specimens or antituberculosis susceptibility testing.

Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management.

We evaluated the COBAS AMPLICOR (CA) PCR system (Roche Diagnostic Systems) designed for automated PCR amplification and detection of nucleic acids from infectious agents in clinical samples. The Roche AMPLICOR microwell plate (MWP) PCR was the reference method. CA amplifies target nucleic acid, captures the biotinylated amplification products by using magnetic particles coated with specific oligonucleotide probes, and detects the bound products colorimetrically. For Mycobacterium tuberculosis, the correlation of the results of CA tests with those of MWP tests was 100% with 230 samples, including 20 culture-positive samples. For hepatitis C virus, the correlation was 100% with 214 samples, including 60 positive samples. MultiPlex CA analysis of 199 cervical specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and the internal control gave 100% concordance. These samples included 19 C. trachomatis and 3 N. gonorrhoeae culture-positive samples. Overall, the agreement between PCR methods for all 842 comparisons was 100%. Compared with culture, the sensitivities of the assays for C. trachomatis and M tuberculosis were > or = 95%. After spiking alternating amplification tubes in the CA system with 10(14) copies of the Chlamydia amplicon per ml, we were unable to demonstrate any carryover cross-contamination of negative samples. Using the criteria of the College of American Pathologists workload recording method, we found that the total hands-on time to produce CA PCR results was 4.4, 7.9, and 3.3 min for M. tuberculosis, hepatis C virus, and the MultiPlexed assay for chlamydia plus gonorrhea and an internal control, respectively. The CA system brings true PCR automation to laboratories. In addition to the accuracy of automated results, the CA system provides labor savings, provides containment of the amplification and detection components of PCR, and supports both MultiPlex amplification and sequential algorithm (ReFlex) detection of analytes.

Evaluation of Amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens.

We evaluated the Amplicor PCR assay (Roche Molecular Systems, Branchburg, N.J.) for direct detection of Mycobacterium tuberculosis in sputum. A total of 532 specimens from 270 patients were decontaminated and stored at 4 or -75 degrees C until assayed by PCR. This assay used three-step sample preparation, biotinylated primer pairs, AmpErase, and a microtiter format for amplicon capture and detection. Amplicor PCR results were compared with clinical history, culture from a Lowenstein-Jensen slant, and results from the BACTEC TB-460 system. Eighty-seven cultures from 15 patients grew M. tuberculosis; of these, 83 (95%) were positive with the Amplicor PCR test. The false negatives were most likely due to sample variation and inhibitors. Of the 445 specimens from which M. tuberculosis was not isolated, 428 (96%) were negative with the Amplicor PCR test. Of the 17 M. tuberculosis culture-negative, Amplicor-positive specimens, 15 were reclassified as true positives because previous cultures grew M. tuberculosis. Of the 445 specimens which did not grow M. tuberculosis, Mycobacterium spp. other than M. tuberculosis were isolated from 150 specimens. Three of these 150 specimens were Amplicor positive; two were from a patient with a history of tuberculosis, and one specimen gave a false-positive result. We do not feel that this represents cross-reactivity, because repeated Amplicor testing of the isolate gave negative results. The microtiter plate has 96 wells. Allowing for six controls, 90 decontaminated specimens can be tested by one technologist in 7.5 h. This PCR assay took 7.5 h to complete and is a sensitive and specific, rapid method for the direct detection of M. tuberculosis from sputum.

Does the use of lidocaine affect the culture of percutaneous bone biopsy specimens obtained to diagnose osteomyelitis? An in vitro and in vivo study.

OBJECTIVE: When percutaneous bone biopsy is done by radiologists, local anesthetics such as lidocaine are routinely used. Although percutaneous bone biopsy of neoplasms is well accepted, it has been suggested that this procedure not be used to diagnose osteomyelitis because of a reported bactericidal effect of lidocaine and related drugs on certain organisms. The purposes of this study were to determine if lidocaine is bactericidal in vitro and to determine if it has an effect on the culture of bacteria in specimens obtained by percutaneous bone biopsy in vivo. SUBJECTS AND METHODS: The minimal inhibitory concentration and minimal bactericidal concentration of 1% lidocaine hydrochloride (10 mg/ml preserved with methylparaben) were determined in vitro for seven bacteria known to be frequent causes of osteomyelitis by using conventional clinical microbiologic methods. Percutaneous core bone biopsy for suspected osteomyelitis was done in 28 patients: 21 with and seven without the use of lidocaine. Sites sampled included vertebrae (14); calcanei, pubis, and ischia (two each); and intervertebral disks (eight). Six of the 21 patients who had percutaneous biopsy with lidocaine also had an open surgical biopsy without lidocaine. The results of cultures of the specimens were compared. Histologic evaluation and radiographic follow-up were used to identify false-negative results. RESULTS: The minimal inhibitory and the minimal bactericidal concentrations, respectively, of lidocaine (in milligrams per milliliter) were as follows: Klebsiella pneumoniae, 5.0 and > 5.0; group B streptococci, 2.5 and 5.0; Staphylococcus aureus, > 5.0; and > 5.0; methicillin-resistant S. aureus, > 5.0 and > 5.0; Escherichia coli, 2.5 and > 5.0; Pseudomonas aeruginosa, 5.0 and 5.0; Salmonella species, 5.0 and > 5.0. We found no difference in bacterial growth and the number of false-negative results between patients who had biopsies with and those who had biopsies without lidocaine. Fifty percent of patients who had growth on cultures of specimens from percutaneous biopsies done with lidocaine had no growth on cultures of specimens from surgical biopsies done without lidocaine. This likely occurred because the surgical specimens were not obtained under cross-sectional imaging guidance. CONCLUSION: Up to a 50% mixture of lidocaine has no significant effect in vitro on the bacterial growth of the seven organisms that cause osteomyelitis most frequently, and no inhibitory effect on bacterial growth was seen in biopsies done with lidocaine in vivo. The inhibitory effect of lidocaine therefore occurs at a greater concentration than is used clinically. We conclude that lidocaine used for biopsy does not interfere with the diagnosis of osteomyelitis.

Incidence of bacteremia associated with chorionic villus sampling.

OBJECTIVE: To assess the frequency of transient bacteremia among women undergoing transabdominal and transcervical chorionic villus sampling (CVS). METHODS: One hundred fourteen women undergoing CVS consented to participate in a university review board-approved study protocol. Exclusion criteria included known cardiac valve anomaly or replacement (or other prosthetic) and antibiotic use within the preceding 21 days. Blood cultures (aerobic and anaerobic) were drawn by a single operator on all patients, before CVS and within 15 minutes after completing CVS. Either the catheter tip or needle tip aspirate from each procedure was also sent for culture. RESULTS: Post-procedure bacteremia was detected in two (1.8%) of the patients undergoing CVS. These two patients both had their procedures performed transcervically, resulting in a 4.1% (two of 49) bacteremia rate after transcervical CVS, compared to none (zero of 65) in the transabdominal group (P = .36). The incidence of positive cultures from sampling instruments was also higher in the transcervical group (16.3 versus 0%; P = .003), but did not result in comparable rates of bacteremia among patients with positive instrument cultures. CONCLUSIONS: In this study, CVS was associated with a low rate of bacteremia, regardless of the procedure route. Recommendations for antibiotic prophylaxis in women with abnormal cardiac valves should parallel those for spontaneous vaginal delivery and other comparable genitourinary procedures.