Validation of a qualitative real-time PCR assay for the detection of Candida auris in hospital inpatient screening.

Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1-2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings. IMPORTANCE: This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening.

Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA.

Background: In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods: We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results: Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions: These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.

Clinical Outcomes of Submicroscopic Infections and Correlates of Protection of VAR2CSA Antibodies in a Longitudinal Study of Pregnant Women in Colombia.

Malaria in pregnancy can cause serious adverse outcomes for the mother and the fetus. However, little is known about the effects of submicroscopic infections (SMIs) in pregnancy, particularly in areas where Plasmodium falciparum and Plasmodium vivax cocirculate. A cohort of 187 pregnant women living in Puerto Libertador in northwest Colombia was followed longitudinally from recruitment to delivery. Malaria was diagnosed by microscopy, reverse transcription-quantitative PCR (RT-qPCR), and placental histopathology. Gestational age, hemoglobin concentration, VAR2CSA-specific IgG levels, and adhesion-blocking antibodies were measured during pregnancy. Statistical analyses were performed to evaluate the impact of SMIs on birth weight and other delivery outcomes. Twenty-five percent of women (45/180) were positive for SMIs during pregnancy. Forty-seven percent of infections (21/45) were caused by P. falciparum, 33% were caused by P. vivax, and 20% were caused by mixed Plasmodium spp. Mixed infections of P. falciparum and P. vivax were associated with lower gestational age at delivery (P = 0.0033), while other outcomes were normal. Over 60% of women had antibodies to VAR2CSA, and there was no difference in antibody levels between those with and without SMIs. The anti-adhesion function of these antibodies was associated with protection from SMI-related anemia at delivery (P = 0.0086). SMIs occur frequently during pregnancy, and while mixed infections of both P. falciparum and P. vivax were not associated with a decrease in birth weight, they were associated with significant risk of preterm birth. We propose that the lack of adverse delivery outcomes is due to functional VAR2CSA antibodies that can protect pregnant women from SMI-related anemia.

Molecular point-of-care devices for the diagnosis of infectious diseases in resource-limited settings - A review of the current landscape, technical challenges, and clinical impact.

Molecular point-of-care (POC) tests offer high sensitivity, rapid turnaround times, relative ease of use, and the convenience of laboratory-grade testing in the absence of formal laboratory spaces and equipment, making them appealing options for infectious disease diagnosis in resource-limited settings. In this review, we discuss the role and potential of molecular POC tests in resource-limited settings and their associated logistical challenges. We discuss U.S. Food and Drug Administration approval, Clinical Laboratory Improvement Amendments complexity levels, and the REASSURED criteria as a starting point for assessing options currently available inside and outside of the United States. We then present POC tests currently in research and development phases that have potential for commercialization and implementation in limited-resource settings. Finally, we review published studies that have assessed the clinical impact of molecular POC testing in limited- and moderate-resource settings.

Standardization of SARS-CoV-2 Cycle Threshold Values: Multisite Investigation Evaluating Viral Quantitation across Multiple Commercial COVID-19 Detection Platforms.

The demand for testing during the coronavirus disease 2019 (COVID-19) pandemic has resulted in the production of several different commercial platforms and laboratory-developed assays for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has created several challenges, including, but not limited to, the standardization of diagnostic testing, utilization of cycle threshold (CT) values for quantitation and clinical interpretation, and data harmonization. Using reference standards consisting of a linear range of SARS-CoV-2 concentrations quantitated by viral culture-based methods and droplet digital PCR, we investigated the commutability and standardization of SARS-CoV-2 quantitation across different laboratories in the United States. We assessed SARS-CoV-2 CT values generated on multiple reverse transcription-PCR (RT-PCR) platforms and analyzed PCR efficiencies, linearity, gene targets, and CT value agreement. Our results demonstrate the inappropriateness of using SARS-CoV-2 CT values without established standards for viral quantitation. Further, we emphasize the importance of using reference standards and controls validated to independent assays, to compare results across different testing platforms and move toward better harmonization of COVID-19 quantitative test results. IMPORTANCE From the onset of the COVID-19 pandemic, the demand for SARS-CoV-2 testing has resulted in an explosion of analytical tests with very different approaches and designs. The variability in testing modalities, compounded by the lack of available commercial reference materials for standardization early in the pandemic, has led to several challenges regarding data harmonization for viral quantitation. In this study, we assessed multiple commercially available RT-PCR platforms across different laboratories within the United States using standardized reference materials characterized by viral culture methods and droplet digital PCR. We observed variability in the results generated by different instruments and laboratories, further emphasizing the importance of utilizing validated reference standards for quantitation, to better harmonize SARS-CoV-2 test results.

Likelihood of hospitalization for a chronic respiratory condition following pediatric infection with enterovirus and rhinovirus strains.

Background: Rhino-enteroviruses, particularly enterovirus strain D68 (EV-D68), have been associated with severe respiratory distress in children. The goal of this study was to compare the long-term outcomes of children with EV-D68 infection to that of children with other enterovirus / rhinovirus. Methods: Nasopharyngeal swabs from 174 children presenting with respiratory distress were tested by PCR for respiratory viruses. The primary outcome was diagnosis of a chronic respiratory condition within the follow-up period. Admission to intensive care, and length of stay were recorded. Odds ratios were determined using multinomial logistic regression. Results: During 5 years of follow-up, the crude odds of diagnosis with a chronic respiratory condition were significantly more likely in EV-D68 cases (OR: 1.95, 95% CI: 1.02, 3.82), but failed to remain significant after adjusting for a past history of asthma. Upon admission for a primary concern of asthma, length of stay both in hospital and intensive care were significantly longer in EV-D68 cases (OR: 2.10 [95% CI: 1.56, 2.82, p < 0.001]) and (OR: 5.18 [95% CI: 1.90, 6.28, p < 0.001]), respectively. After adjustment for a history of asthma, EV-D68 cases had significantly longer length of stay in hospital, admitted for 1.94 days for each day that controls were admitted (95% CI: 1.40, 2.68). In admissions to intensive care, EV-D68 cases spent 2.74 days for each day of admission in controls (95% CI: 1.62, 4.97, p < 0.001). Conclusions: Ours is first study to assess prognostic respiratory outcomes of patients infected with EV-D68 in childhood. Our study finds that EV-D68 cases were significantly more likely be hospitalized for longer than other enterovirus/rhinovirus controls in subsequent admissions for respiratory distress. Need for intensive care was significantly longer in EV-D68 infections. Our next steps will be validation in a larger sample size.

Effect of Plasmodium Infection during Pregnancy on Passive Neonatal Immunity against Tetanus Toxoid and Rotavirus.

Passive immunity acquired through transplacental IgG transport is essential to protect infants against pathogens as childhood vaccination programs begins. Diarrhea caused by rotavirus and neonatal tetanus are common and potentially fatal childhood infections that can be prevented by transplacental IgG. However, it is not known whether maternal infections in pregnancy can reduce the transfer of these antibodies to the fetus. This study evaluated the effect of submicroscopic Plasmodium infection during pregnancy on the transfer of maternal IgG antibodies against rotavirus (anti-RV) and tetanus toxoid (anti-TT) to newborns of pregnant women residing in Puerto Libertador and Tierralta, Colombia. Expression of different immune mediators and levels of IgG against rotavirus and tetanus toxoid were quantified in pregnant women with and without Plasmodium infection during pregnancy. Submicroscopic infection at the time of delivery was associated with a cord-to-maternal ratio (CMR) > 1 for anti-RV and < 1 for anti-TT IgG, as well as with an increase in the expression of immune mediators of inflammation (IFN-γ), anti-inflammation (IL-10, TGF-ß), and regulation (FoxP3, CTLA-4). When compared by species, these findings (CMR > 1 for anti-RV and < 1 for anti-TT IgG) were conserved in submicroscopic Plasmodium vivax infections at delivery. The impact of Plasmodium infections on neonatal susceptibility to other infections warrants further exploration.

A sensitive species-specific reverse transcription real-time PCR method for detection of Plasmodium falciparum and Plasmodium vivax.

As the global burden of malaria decreases and countries strive towards disease elimination, there is a greater demand for sensitive diagnostics to target the submicroscopic reservoir of infection. We describe here a sensitive species-specific RT-qPCR method to differentiate between Plasmodium falciparum and P. vivax infections at the submicroscopic level. With amplification of the 18S rRNA genes from total nucleic acids (both DNA and RNA), we discern P. falciparum and P. vivax with a limit of detection of 10 parasites/mL and 18 copies/µL, respectively. This assay was validated with 519 blood samples, negative by thick-smear, from febrile and asymptomatic cohorts from Colombia. These results were directly compared to a qPCR-based method (DNA only) as the gold standard. Of the samples from patients who presented with fever (n = 274), 34 infections were identified by RT-qPCR (16 P. falciparum, 15 P. vivax, and 3 mixed), of which only 10 infections were identified at the species level by qPCR. Within the asymptomatic cohort (n = 245), 13 infections were identified by RT-qPCR (3 P. falciparum, 3 P. vivax, and 7 mixed), whereas the species for only one infection was determined by qPCR. We conclude that this species-specific RT-qPCR method provides a more sensitive tool for species identification compared to DNA based qPCR methods.

Impact of Malaria in Pregnancy as Latin America Approaches Elimination.

In Latin America, four million pregnancies are at risk of malaria annually, but malaria in pregnancy is largely overlooked. As countries progress toward malaria elimination, targeting reservoirs of transmission is a priority. Pregnant women are an important risk group because they harbor asymptomatic infections and dormant liver stages of Plasmodium vivax that cause relapses. Of significant concern is the discovery that most infections in pregnant women fail to be detected by routine diagnostics. We review here recent findings on malaria in pregnancy within Latin America. We focus on the Amazon basin and Northwest Colombia, areas that harbor the greatest burden of malaria, and propose that more sensitive diagnostics and active surveillance at antenatal clinics will be necessary to eliminate malaria from these final frontiers.