Assessment of subpatent Plasmodium infection in northwestern Ethiopia.

BACKGROUND: Ethiopia has set a goal for malaria elimination by 2030. Low parasite density infections may go undetected by conventional diagnostic methods (microscopy and rapid diagnostic tests) and their contribution to malaria transmission varies by transmission settings. This study quantified the burden of subpatent infections from samples collected from three regions of northwest Ethiopia. METHODS: Sub-samples of dried blood spots from the Ethiopian Malaria Indicator Survey 2015 (EMIS-2015) were tested and compared using microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (nPCR) to determine the prevalence of subpatent infection. Paired seroprevalence results previously reported along with gender, age, and elevation of residence were explored as risk factors for Plasmodium infection. RESULTS: Of the 2608 samples collected, the highest positive rate for Plasmodium infection was found with nPCR 3.3% (95% CI 2.7-4.1) compared with RDT 2.8% (95% CI 2.2-3.5) and microscopy 1.2% (95% CI 0.8-1.7). Of the nPCR positive cases, Plasmodium falciparum accounted for 3.1% (95% CI 2.5-3.8), Plasmodium vivax 0.4% (95% CI 0.2-0.7), mixed P. falciparum and P. vivax 0.1% (95% CI 0.0-0.4), and mixed P. falciparum and Plasmodium malariae 0.1% (95% CI 0.0-0.3). nPCR detected an additional 30 samples that had not been detected by conventional methods. The majority of the nPCR positive cases (61% (53/87)) were from the Benishangul-Gumuz Region. Malaria seropositivity had significant association with nPCR positivity [adjusted OR 10.0 (95% CI 3.2-29.4), P < 0.001]. CONCLUSION: Using nPCR the detection rate of malaria parasites increased by nearly threefold over rates based on microscopy in samples collected during a national cross-sectional survey in 2015 in Ethiopia. Such subpatent infections might contribute to malaria transmission. In addition to strengthening routine surveillance systems, malaria programmes may need to consider low-density, subpatent infections in order to accelerate malaria elimination efforts.

Multiplex serology demonstrate cumulative prevalence and spatial distribution of malaria in Ethiopia.

BACKGROUND: Measures of malaria burden using microscopy and rapid diagnostic tests (RDTs) in cross-sectional household surveys may incompletely describe the burden of malaria in low-transmission settings. This study describes the pattern of malaria transmission in Ethiopia using serological antibody estimates derived from a nationwide household survey completed in 2015. METHODS: Dried blood spot (DBS) samples were collected during the Ethiopian Malaria Indicator Survey in 2015 from malarious areas across Ethiopia. Samples were analysed using bead-based multiplex assays for IgG antibodies for six Plasmodium antigens: four human malaria species-specific merozoite surface protein-1 19kD antigens (MSP-1) and Apical Membrane Antigen-1 (AMA-1) for Plasmodium falciparum and Plasmodium vivax. Seroprevalence was estimated by age, elevation and region. The seroconversion rate was estimated using a reversible catalytic model fitted with maximum likelihood methods. RESULTS: Of the 10,278 DBS samples available, 93.6% (9622/10,278) had valid serological results. The mean age of participants was 15.8 years and 53.3% were female. National seroprevalence for antibodies to P. falciparum was 32.1% (95% confidence interval (CI) 29.8-34.4) and 25.0% (95% CI 22.7-27.3) to P. vivax. Estimated seroprevalences for Plasmodium malariae and Plasmodium ovale were 8.6% (95% CI 7.6-9.7) and 3.1% (95% CI 2.5-3.8), respectively. For P. falciparum seroprevalence estimates were significantly higher at lower elevations (< 2000 m) compared to higher (2000-2500 m) (aOR 4.4; p < 0.01). Among regions, P. falciparum seroprevalence ranged from 11.0% (95% CI 8.8-13.7) in Somali to 65.0% (95% CI 58.0-71.4) in Gambela Region and for P. vivax from 4.0% (95% CI 2.6-6.2) in Somali to 36.7% (95% CI 30.0-44.1) in Amhara Region. Models fitted to measure seroconversion rates showed variation nationally and by elevation, region, antigen type, and within species. CONCLUSION: Using multiplex serology assays, this study explored the cumulative malaria burden and regional dynamics of the four human malarias in Ethiopia. High malaria burden was observed in the northwest compared to the east. High transmission in the Gambela and Benishangul-Gumuz Regions and the neglected presence of P. malariae and P. ovale may require programmatic attention. The use of a multiplex assay for antibody detection in low transmission settings has the potential to act as a more sensitive biomarker.

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. IMPORTANCE: Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions.

The murine gammaherpesvirus immediate-early Rta synergizes with IRF4, targeting expression of the viral M1 superantigen to plasma cells.

MHV68 is a murine gammaherpesvirus that infects laboratory mice and thus provides a tractable small animal model for characterizing critical aspects of gammaherpesvirus pathogenesis. Having evolved with their natural host, herpesviruses encode numerous gene products that are involved in modulating host immune responses to facilitate the establishment and maintenance of lifelong chronic infection. One such protein, MHV68 M1, is a secreted protein that has no known homologs, but has been shown to play a critical role in controlling virus reactivation from latently infected macrophages. We have previous demonstrated that M1 drives the activation and expansion of Vß4+ CD8+ T cells, which are thought to be involved in controlling MHV68 reactivation through the secretion of interferon gamma. The mechanism of action and regulation of M1 expression are poorly understood. To gain insights into the function of M1, we set out to evaluate the site of expression and transcriptional regulation of the M1 gene. Here, using a recombinant virus expressing a fluorescent protein driven by the M1 gene promoter, we identify plasma cells as the major cell type expressing M1 at the peak of infection in the spleen. In addition, we show that M1 gene transcription is regulated by both the essential viral immediate-early transcriptional activator Rta and cellular interferon regulatory factor 4 (IRF4), which together potently synergize to drive M1 gene expression. Finally, we show that IRF4, a cellular transcription factor essential for plasma cell differentiation, can directly interact with Rta. The latter observation raises the possibility that the interaction of Rta and IRF4 may be involved in regulating a number of viral and cellular genes during MHV68 reactivation linked to plasma cell differentiation.

Identification of alternative transcripts encoding the essential murine gammaherpesvirus lytic transactivator RTA.

UNLABELLED: The essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-α/ßR-/- mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-α). 5' rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-α treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-α/ßR-/- mice with the G50DblKo mutant virus demonstrated partial rescue of (i) acute virus replication in the lungs, (ii) establishment of latency, and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlights the complex mechanisms involved in controlling expression of RTA, likely reflecting time-dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression. IMPORTANCE: The viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases.

Murine gammaherpesvirus 68 reactivation from B cells requires IRF4 but not XBP-1.

UNLABELLED: Gammaherpesviruses display tropism for B cells and, like all known herpesviruses, exhibit distinct lytic and latent life cycles. One well-established observation among members of the gammaherpesvirus family is the link between viral reactivation from latently infected B cells and plasma cell differentiation. Importantly, a number of studies have identified a potential role for a CREB/ATF family member, X-box binding protein 1 (XBP-1), in trans-activating the immediate early BZLF-1 or BRLF1/gene 50 promoters of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), respectively. XBP-1 is required for the unfolded protein response and has been identified as a critical transcription factor in plasma cells. Here, we demonstrate that XBP-1 is capable of trans-activating the murine gammaherpesvirus 68 (MHV68) RTA promoter in vitro, consistent with previous observations for EBV and KSHV. However, we show that in vivo there does not appear to be a requirement for XBP-1 expression in B cells for virus reactivation. The MHV68 M2 gene product under some experimental conditions plays an important role in virus reactivation from B cells. M2 has been shown to drive B cell differentiation to plasma cells, as well as interleukin-10 (IL-10) production, both of which are dependent on M2 induction of interferon regulatory factor 4 (IRF4) expression. IRF4 is required for plasma cell differentiation, and consistent with a role for plasma cells in MHV68 reactivation from B cells, we show that IRF4 expression in B cells is required for efficient reactivation of MHV68 from splenocytes. Thus, the latter analyses are consistent with previous studies linking plasma cell differentiation to MHV68 reactivation from B cells. The apparent independence of MHV68 reactivation from XBP-1 expression in plasma cells may reflect redundancy among CREB/ATF family members or the involvement of other plasma cell-specific transcription factors. Regardless, these findings underscore the importance of in vivo studies in assessing the relevance of observations made in tissue culture models. IMPORTANCE: All known herpesviruses establish a chronic infection of their respective host, persisting for the life of the individual. A critical feature of these viruses is their ability to reactivate from a quiescent form of infection (latency) and generate progeny virus. In the case of gammaherpesviruses, which are associated with the development of lymphoproliferative disorders, including lymphomas, reactivation from latently infected B lymphocytes occurs upon terminal differentiation of these cells to plasma cells-the cell type that produces antibodies. A number of studies have linked a plasma cell transcription factor, XBP-1, to the induction of gammaherpesvirus reactivation, and we show here that indeed in tissue culture models this cellular transcription factor can trigger expression of the murine gammaherpesvirus gene involved in driving virus reactivation. However, surprisingly, when we examined the role of XBP-1 in the setting of infection of mice-using mice that lack a functional XBP-1 gene in B cells-we failed to observe a role for XBP-1 in virus reactivation. However, we show that another cellular factor essential for plasma cell differentiation, IRF4, is critical for virus reactivation. Thus, these studies point out the importance of studies in animal models to validate findings from studies carried out in cell lines passaged in vitro.

Antibody dynamics in children with first or repeat Plasmodium falciparum infections.

Immunoglobulin (Ig) production during and after infection with Plasmodium parasites is one of the greatest adaptive immune defenses the human host has against this parasite. Infection with P. falciparum has been shown to induce different B cell maturation responses dependent upon the age of the patient, number of previous exposures, and severity of the disease. Described here are dynamics of Ig responses to a panel of 32 P. falciparum antigens by patients followed for 42 days and classified individuals as showing characteristics of an apparent first P. falciparum infection (naïve) or a repeat exposure (non-naïve). Six parameters were modeled to characterize the dynamics of IgM, IgG1, IgG3, and IgA for these two exposure groups with differences assessed among Ig isotypes/subclasses and unique antigens. Naïve patients had significantly longer periods of time to reach peak Ig titer (range 4-7 days longer) and lower maximum Ig titers when compared with non-naïve patients. Modeled time to seronegativity was significantly higher in non-naïve patients for IgM and IgA, but not for the two IgG subclasses. IgG1 responses to Rh2030, HSP40, and PfAMA1 were at the highest levels for non-naïve participants and may be used to predict previous or nascent exposure by themselves. The analyses presented here demonstrate the differences in the development of the Ig response to P. falciparum if the infection represents a boosting response or a primary exposure. Consistency in Ig isotype/subclasses estimates and specific data for P. falciparum antigens can better guide interpretation of seroepidemiological data among symptomatic persons.

Evaluation of a Multiplex Bead Assay against Single-Target Assays for Detection of IgG Antibodies to SARS-CoV-2.

Serological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected. IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.

Framework for Characterizing Longitudinal Antibody Response in Children After Plasmodium falciparum Infection.

Human Plasmodium infection produces a robust adaptive immune response. Time courses for 104 children followed for 42 days after initiation of Plasmodium falciparum chemotherapy were assayed for antibody levels to the five isotypes of human immunoglobulins (Ig) and 4 subclasses of IgG for 32 P. falciparum antigens encompassing all 4 parasite stages of human infection. IgD and IgE against these antigens were undetectable at 1:100 serum concentration, but other Ig isotypes and IgG subclasses were consistently observed against all antigens. Five quantitative parameters were developed to directly compare Ig response among isotypes and antigens: Cmax, maximum antibody level; ΔC, difference between Cmax and the antibody level at Day 0; tmax, time in days to reach Cmax; t1/2, Ig signal half-life in days; tneg, estimated number of days until complete loss of Ig signal. Classical Ig patterns for a bloodborne pathogen were seen with IgM showing early tmax and IgG production highest among Ig isotypes. However, some unexpected trends were observed such as IgA showing a biphasic pattern for many antigens. Variability among these dynamics of Ig acquisition and loss was noted for different P. falciparum antigens and able to be compared both quantitatively and statistically. This parametrization methodology allows direct comparison of Ig isotypes produced against various Plasmodium antigens following malaria infection, and the same methodology could be applied to other longitudinal serologic studies from P. falciparum or different pathogens. Specifically for P. falciparum seroepidemiological studies, reliable and quantitative estimates regarding the IgG dynamics in human populations can better optimize modeling efforts for serological outputs.

CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice.

Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vß4+ CD8+ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8+ T cells and MHV68 epitope specific CD8+ T cells to the lungs-despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vß4+ CD8+ T cell expansion, eliminated MHV68-induced fibrosis-further implicating the activated Vß4+ CD8+ T cell population in the induction of fibrosis. We further addressed the role that CD8+ T cells play in the induction of fibrosis by depleting CD8+ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vß4+ CD8+ T cells as mediators of fibrotic disease in IFNγR-/- mice.