Evaluation of the Performance of Ortho T. cruzi ELISA Test System for the Detection of Antibodies to Trypanosoma cruzi.

The serologic diagnosis of chronic Chagas disease, caused by infection with the parasite Trypanosoma cruzi, is challenging and lacks a gold-standard assay. To overcome the problem, CDC uses an algorithm that uses two tests on different platforms and applies a third test as a tiebreaker. The Ortho T. cruzi ELISA Test System from Ortho Diagnostics was cleared by FDA for clinical diagnosis usage. We evaluated this test against the CDC algorithm for chronic Chagas disease. We tested several sets of serum specimens: 104 specimens tested positive for T. cruzi specific antibody and 283 (including 30 specimens positive for antibody to Leishmania spp.) tested negative based on the current CDC chronic T. cruzi infection diagnostic testing algorithm. Concordance of the Ortho T. cruzi ELISA Test System with the CDC algorithm result was 90% (95% CI 87 to 93%) overall and 92% (95% CI 89 to 95%) when excluding Leishmania spp. antibody positive specimens. The cross-reactivity of the Ortho T. cruzi ELISA Test System was 37% to Leishmania spp. serologically positive specimens, 1% to specimens from patients diagnosed with other parasitic infections, and 0% against specimens from a US noninfected population. In conclusion, the Ortho T. cruzi ELISA Test System compares well against the CDC diagnostic algorithm for chronic Chagas disease. The availability of this FDA-cleared assay will improve the chronic Chagas disease diagnosis.

Evidence of likely autochthonous Chagas disease in the southwestern United States: A case series of Trypanosoma cruzi seropositive blood donors.

BACKGROUND: Chagas disease is a parasitic infection that can insidiously cause non-ischemic cardiomyopathy. Given the largely silent nature of this progressive disease, asymptomatic blood donors pose potential blood transfusion risk. Blood donation screening has become an unintentional form of Chagas disease surveillance, with thousands of new cases identified since national surveillance was initiated in 2007. STUDY DESIGN AND METHODS: We recruited T. cruzi-positive blood donors identified from California and Arizona blood centers for confirmatory blood screening and assessment of lifetime infection risk. RESULTS: Among eight suspected cases, we identified four confirmed US autochthonous infections. The current manuscript details the transmission sources, healthcare-seeking behaviors post-blood donation resulting, and clinical course of disease among persons without any history of travel to endemic Latin American countries. DISCUSSION: This manuscript presents four additional US-acquired Chagas disease cases and identifies an opportunity for blood centers to assist in confronting barriers surrounding Chagas disease in the US.

Trypanosoma cruzi in Nonischemic Cardiomyopathy Patients, Houston, Texas, USA.

To investigate possible cardiac manifestations of Chagas disease, we tested 97 Latinx patients with nonischemic cardiomyopathy in Houston, Texas, USA, for Trypanosoma cruzi infection. We noted a high prevalence of underdiagnosed infection and discrepant results in clinical diagnostic assays. Latinx cardiac patients in the United States would benefit from laboratory screening for T. cruzi infection.

Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Investigation of a case of suspected transfusion-transmitted malaria.

BACKGROUND: Transfusion-transmitted malaria (TTM) is a rare occurrence with serious consequences for the recipient. A case study is presented as an example of best practices for conducting a TTM investigation. CASE REPORT: A 15-year-old male with a history of sickle cell disease developed fever after a blood transfusion. He was diagnosed with Plasmodium falciparum malaria and was successfully treated. The American Red Cross, New York State Department of Health, and the Centers for Disease Control and Prevention investigated the eight donors who provided components to the transfusion. The investigation to identify a malaria-positive donor included trace back of donors, serologic methods to identify donor(s) with a history of malaria exposure, polymerase chain reaction (PCR) testing, microsatellite analysis to identify the parasite in a donor and match its genotype to the parasite in the recipient, and reinterview of all donors to clarify malaria risk factors. RESULTS: One donor had evidence of infection with P. falciparum by PCR, elevated antibody titers, and previously undisclosed malaria risk factors. Reinterview revealed that the donor immigrated to the United States from Togo just short of 3 years before the blood donation. The donor was treated for asymptomatic low parasitemia infection. CONCLUSION: This investigation used standard procedures for investigating TTM but also demonstrated the importance of applying sensitive laboratory techniques to identify the infected donor, especially a donor with asymptomatic infection with low parasitemia. Repeat interview of all donors identified as having contributed to the transfused component provides complementary epidemiologic information to confirm the infected donor.

Reactivation of Chagas disease among heart transplant recipients in the United States, 2012-2016.

BACKGROUND: Heart transplantation has been shown to be a safe and effective intervention for progressive cardiomyopathy from chronic Chagas disease. However, in the presence of the immunosuppression required for heart transplantation, the likelihood of Chagas disease reactivation is significant. Reactivation may cause myocarditis resulting in allograft dysfunction and the rapid onset of congestive heart failure. Reactivation rates have been well documented in Latin America; however, there is a paucity of data regarding the risk in non-endemic countries. METHODS: We present our experience with 31 patients with chronic Chagas disease who underwent orthotopic heart transplantation in the United States from 2012 to 2016. Patients were monitored following a standard schedule. RESULTS: Of the 31 patients, 19 (61%) developed evidence of reactivation. Among the 19 patients, a majority (95%) were identified by laboratory monitoring using polymerase chain reaction testing. One patient was identified after the onset of clinical symptoms of reactivation. All subjects with evidence of reactivation were alive at follow-up (median: 60 weeks). CONCLUSIONS: Transplant programs in the United States are encouraged to implement a monitoring program for heart transplant recipients with Chagas disease. Our experience using a preemptive approach of monitoring for Chagas disease reactivation was effective at identifying reactivation before symptoms developed.

Likely Autochthonous Transmission of Trypanosoma cruzi to Humans, South Central Texas, USA.

Chagas disease, caused by Trypanosoma cruzi, is a major neglected tropical disease affecting the Americas. The epidemiology of this disease in the United States is incomplete. We report evidence of likely autochthonous vectorborne transmission of T. cruzi and health outcomes in T. cruzi-seropositive blood donors in south central Texas, USA.

Experimental transfusion-induced Babesia microti infection: dynamics of parasitemia and immune responses in a rhesus macaque model.

BACKGROUND: Babesiosis is an emerging tick-borne infection in humans. The increasing numbers of reported cases of transfusion-associated babesiosis (TAB), primarily caused by Babesia microti, represents a concern for the safety of the US blood supply. STUDY DESIGN AND METHODS: This study investigated kinetics of parasitemia and innate immune responses and dynamics of antibody responses during B. microti infection in rhesus macaques (RMs) using blood smears, quantitative polymerase chain reaction (qPCR), flow cytometry, and indirect fluorescent antibody testing. A total of six monkeys were transfused with either hamster or monkey-passaged B. microti-infected red blood cells (two and four monkeys, respectively) simulating TAB. RESULTS: The prepatent period in monkeys inoculated with hamster-passaged B. microti was 35 days compared with 4 days in monkeys transfused with monkey-passaged B. microti; the latter monkeys also had markedly higher parasitemia levels. The duration of the window period from the first detected parasitemia by qPCR analysis to the first detected antibody response ranged from 10 to 17 days. Antibody responses fluctuated during the course of the infection. Innate responses assessed by the frequencies of monocytes and activated B cells correlated with the kinetics and magnitude of parasitemia. On Day 14, additional activation peaks were noted for CD14+CD16+ and CD14-CD16+ monocytes and for CD11c+ myeloid dendritic cells, but only in animals transfused with monkey-passaged B. microti. Parasitemia persisted in these immunocompetent animals, similar to human infection. CONCLUSION: The results suggest that transfusion-associated transmission of B. microti leads to rapid onset of parasitemia (Day 4) in RMs, detectable antibody response 14 days later, and persistent parasitemia.

Raccoon Roundworm Infection Associated with Central Nervous System Disease and Ocular Disease - Six States, 2013-2015.

Baylisascaris procyonis, predominantly found in raccoons, is a ubiquitous roundworm found throughout North America. Although raccoons are typically asymptomatic when infected with the parasite, the larval form of Baylisascaris procyonis can result in fatal human disease or severe neurologic outcomes if not treated rapidly. In the United States, Baylisascaris procyonis is more commonly enzootic in raccoons in the midwestern and northeastern regions and along the West Coast (1). However, since 2002, infections have been documented in other states (Florida and Georgia) and regions (2). Baylisascariasis is not a nationally notifiable disease in the United States, and little is known about how commonly it occurs or the range of clinical disease in humans. Case reports of seven human baylisascariasis cases in the United States diagnosed by Baylisascaris procyonis immunoblot testing at CDC are described, including review of clinical history and laboratory data. Although all seven patients survived, approximately half were left with severe neurologic deficits. Prevention through close monitoring of children at play, frequent handwashing, and clearing of raccoon latrines (communal sites where raccoons defecate) are critical interventions in curbing Baylisascaris infections. Early treatment of suspected cases is critical to prevent permanent sequelae.

Fatal Case of Polymicrobial Meningitis Caused by Cryptococcus liquefaciens and Mycobacterium tuberculosis Complex in a Human Immunodeficiency Virus-Infected Patient.

We describe a fatal case of polymicrobial meningitis in a human immunodeficiency virus-infected patient from Guatemala caused by Cryptococcus liquefaciens and Mycobacterium tuberculosis complex. Central nervous system infections caused concurrently by these species are extremely rare. This is also the first report of disseminated disease caused by C. liquefaciens.

Outbreak of Trichinella spiralis infections associated with a wild boar hunted at a game farm in Iowa.

BACKGROUND: Rates of trichinellosis have declined significantly in the United States due to improved pork production practices and public awareness of the danger of eating raw or undercooked pork. In April 2011, the Minnesota Department of Health received a report of presumptive trichinellosis in a 50-year-old man with a history of wild boar consumption. A public health investigation was initiated. METHODS: Medical record reviews and patient and family interviews were conducted. Trichinella species serology was performed on patient and family serum samples, and larval identification was attempted on clinical specimens and meat samples. RESULTS: The index patient harvested a wild boar from an Iowa game farm; he processed the meat after returning home and developed gastrointestinal symptoms 2 days later. Four days after his illness onset, all 5 family members consumed a roast from the boar. The index patient sought healthcare 4 times after illness onset before being definitively diagnosed with trichinellosis. Following initiation of albendazole therapy, the index patient developed atrial fibrillation. One additional family member who processed the raw meat was diagnosed with trichinellosis. Trichinella spiralis larvae were identified in wild boar meat samples. CONCLUSIONS: Trichinellosis has long been recognized as a potential hazard of consuming undercooked wild carnivore meat, and historically has been associated with consumption of pork from domestic swine, but may be unfamiliar to practicing clinicians in the United States. Education of hunters and the broader population on the potential for trichinellosis and the importance of proper handling and cooking meat from wild or free-range animals needs to be reinforced.

In utero nicotine exposure promotes M2 activation in neonatal mouse alveolar macrophages.

BACKGROUND: Maternal smoking in utero has been associated with adverse health outcomes including lower respiratory tract infections in infants and children, but the mechanisms underlying these associations continue to be investigated. We hypothesized that nicotine plays a significant role in mediating the effects of maternal tobacco smoke on the function of the neonatal alveolar macrophage (AM), the resident immune cell in the neonatal lung. METHODS: Primary AMs were isolated at postnatal day 7 from a murine model of in utero nicotine exposure. The murine AM cell line MH-S was used for additional in vitro studies. RESULTS: In utero nicotine increased interleukin-13 and transforming growth factor-ß1 (TGFß1) in the neonatal lung. Nicotine-exposed AMs demonstrated increased TGFß1 and increased markers of alternative activation with diminished phagocytic function. However, AMs from mice deficient in the α7 nicotinic acetylcholine receptor (α7 nAChR) had less TGFß1, reduced alternative activation, and improved phagocytic functioning despite similar in utero nicotine exposure. CONCLUSION: In utero nicotine exposure, mediated in part via the α7 nAChR, may increase the risk of lower respiratory tract infections in neonates by changing the resting state of AM toward alternative activation. These findings have important implications for immune responses in the nicotine-exposed neonatal lung.

A combined transcriptome and proteome survey of malaria parasite liver stages.

For 50 years since their discovery, the malaria parasite liver stages (LS) have been difficult to analyze, impeding their utilization as a critical target for antiinfection vaccines and drugs. We have undertaken a comprehensive transcriptome analysis in combination with a proteomic survey of LS. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to efficiently isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared with other parasite life cycle stages. The analysis revealed approximately 2,000 genes active during LS development, and proteomic analysis identified 816 proteins. A subset of proteins appeared to be expressed in LS only. The data revealed exported parasite proteins and LS metabolic pathways including expression of FASII pathway enzymes. The FASII inhibitor hexachlorophene and the antibiotics, tetracycline and rifampicin, that target the apicoplast inhibited LS development, identifying FASII and other pathways localized in the apicoplast as potential drug targets to prevent malaria infection.

A Bead-Based Assay for the Detection of Antibodies against Trichinella spp. Infection in Humans.

Human trichinellosis can be diagnosed by a combination of medical history, clinical presentation, and laboratory findings, and through detection of anti-Trichinella IgG in the patient's sera. ELISA using excretory-secretory (E/S) antigens of Trichinella spiralis larvae is currently the most used assay to detect Trichinella spp. antibodies. Bead-based assay can detect antibodies to multiple antigens concurrently; the ability to detect antibody to T. spiralis using a bead assay could be useful for diagnosis and surveillance. We developed and evaluated a bead assay to detect and quantify total IgG or IgG4 Trichinella spp. antibodies in human serum using T. spiralis E/S antigens. The sensitivity and specificity of the assay were determined using serum from 110 subjects with a confirmed diagnosis of trichinellosis, 140 subjects with confirmed infections with other tissue-dwelling parasites, 98 human serum samples from residents of the United States with no known history of parasitic infection, and nine human serum samples from residents of Egypt with negative microscopy for intestinal parasites. Sensitivity and specificity were 93.6% and 94.3% for total IgG and 89.2% and 99.2% for IgG4, respectively. Twelve percent of sera from patients with confirmed schistosomiasis reacted with the IgG Trichinella bead assay, as did 11% of sera from patients with neurocysticercosis. The Trichinella spp. bead assay to detect IgG total antibody responses has a similar performance as the Trichinella E/S ELISA. The Trichinella spp. bead assay shows promise as a method to detect trichinellosis with a possibility to be used in multiplex applications.

Antibody epitope repertoire analysis enables rapid antigen discovery and multiplex serology.

The detection of pathogen-specific antibodies remains a cornerstone of clinical diagnostics. Yet, many test exhibit undesirable performance or are completely lacking. Given this, we developed serum epitope repertoire analysis (SERA), a method to rapidly discover conserved, pathogen-specific antigens and their epitopes, and applied it to develop an assay for Chagas disease caused by the protozoan parasite Trypanosoma cruzi. Antibody binding peptide motifs were identified from 28 Chagas repertoires using a bacterial display random 12-mer peptide library and next-generation sequencing (NGS). Thirty-three motifs were selected and mapped to candidate Chagas antigens. In a blinded validation set (n = 72), 30/30 Chagas were positive, 30/30 non-Chagas were negative, and 1/12 Leishmania sp. was positive. After unblinding, a Leishmania cross-reactive epitope was identified and removed from the panel. The Chagas assay exhibited 100% sensitivity (30/30) and specificity (90/90) in a second blinded validation set including individuals with other parasitic infections. Amongst additional epitope repertoires with unknown Chagas serostatus, assay specificity was 99.8% (998/1000). Thus, the Chagas assay achieved a combined sensitivity and specificity equivalent or superior to diagnostic algorithms that rely on three separate tests to achieve high specificity. NGS-based serology via SERA provides an effective approach to discover antigenic epitopes and develop high performance multiplex serological assays.

Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection.

Malaria parasite sporozoites prepare for transmission to a mammalian host by upregulation of UIS (Upregulated in Infectious Sporozoites) genes. A number of UIS gene products are essential for the establishment of the intrahepatocytic niche. However, the factors that regulate the expression of genes involved in gain of infectivity for the liver are unknown. Herein, we show that a conserved Plasmodium sporozoite low-complexity asparagine-rich protein, SAP1 (Sporozoite Asparagine-rich Protein 1), has an essential role in malaria parasite liver infection. Targeted deletion of SAP1 in the rodent malaria parasite Plasmodium yoelii generated mutant parasites that traverse and invade hepatocytes normally but cannot initiate liver-stage development in vitro and in vivo. Moreover, immunizations with Pysap1(-) sporozoites confer long-lasting sterile protection against wild-type sporozoite infection. Strikingly, lack of SAP1 abolished expression of essential UIS genes including UIS3, UIS4 and P52 but not the constitutively expressed genes encoding, among others, sporozoite proteins CSP and TRAP. SAP1 localization to the cell interior but not the nucleus of sporozoites suggests its involvement in a post-transcriptional mechanism of gene expression control. These findings demonstrate that SAP1 is essential for liver infection possibly by functioning as a selective regulator controlling the expression of infectivity-associated parasite effector genes.

Toxoplasma gondii Infection in the United States, 2011-2014.

Toxoplasma gondii can cause severe neurologic and ocular disease when transmitted congenitally and in immunosuppressed persons. Sera collected in the National Health and Nutrition Examination Survey 2011 through 2014 in 13,507 persons ≥ 6 years old were tested for T. gondii immunoglobulin (Ig) G and IgM antibodies, and in those both IgG and IgM antibody positive, for IgG avidity. Overall, 11.14% (95% confidence limits [CL] 9.88%, 12.51%) were seropositive for T. gondii IgG antibody (age-adjusted seroprevalence 10.42% [95% CL 9.19%, 11.76%]); in women aged 15-44 years, the age-adjusted T. gondii IgG seroprevalence was 7.50% (95% CL 6.00%, 9.25%). In multivariable analysis, risk for IgG seropositivity increased with age and was higher in males; persons living below the poverty level; persons with ≤ a high school education compared with those with > a high school education; and non-Hispanic black, Mexican American, and foreign born non-Hispanic white persons compared with U.S.-born non-Hispanic white persons. Overall, 1.16% (95% CL 0.94%, 1.42%) were T. gondii IgM antibody positive and 0.71%, (95% CL 0.54%, 0.92%) were both IgM and IgG antibody positive. In multivariable analysis, the significant risk factors for being both IgM and IgG positive were older age, crowding, and non-U.S. birth origin compared with U.S.-born persons. Among those positive for both IgM and IgG antibody, almost all had high avidity (all women aged 15-44 years had high avidity). Toxoplasma gondii antibody prevalence remains relatively low in the United States, although it is higher in non-U.S.-born persons, males, and some minority and socioeconomically disadvantaged groups.

Anti-tissue remodeling effects of corticosteroids: fluticasone propionate inhibits fibronectin expression in fibroblasts.

OBJECTIVES: Tissue remodeling often accompanies diseases such as COPD that are caused by or aggravated by tobacco exposure. Inhaled or systemic corticosteroids are frequently used for the treatment of these illnesses, and their beneficial effects are often ascribed to their anti-inflammatory properties. However, their role in tissue remodeling remains unclear. This study was designed to evaluate the role of corticosteroids in matrix expression in vitro. DESIGN: We investigated the effects of the corticosteroid fluticasone propionate (FP) on the production of fibronectin by fibroblasts before and after stimulation by nicotine, a plant alkaloid found in tobacco. Fibronectin is an extracellular matrix glycoprotein found elevated in the alveolar lining fluid and airway walls of subjects with obstructive lung disease, and is considered a marker of tissue remodeling after injury. RESULTS: FP, 1 micromol/L, inhibited the expression of fibronectin messenger RNA and protein in unstimulated NIH-3T3 cells and primary lung fibroblasts, as well as in fibroblasts stimulated with nicotine. The inhibitory effect of FP occurred at the level of gene transcription as demonstrated in lung fibroblasts expressing a construct containing the human fibronectin promoter connected to a luciferase reporter gene, but posttranscriptional effects also appeared involved. Electrophoresis mobility gel shift assays revealed that FP inhibited phosphorylation and DNA binding by the cyclic adenosine monophosphate response element binding protein, a transcription factor required for constitutive and nicotine-induced fibronectin expression. CONCLUSIONS: Together, these data suggest that FP could diminish lung tissue remodeling by inhibiting the production of fibronectin in lung fibroblasts.