Self-medication and ILI etiologies among individuals presenting at pharmacies with influenza-like illness: Guatemala City, 2018 influenza season.

OBJECTIVES: We aimed to characterize the proportion of clients presenting to community pharmacies with influenza-like illness (ILI) and the severity of their illness; the proportion with detectable influenza A, influenza B, and other pathogens (i.e., parainfluenza I, II, and III, adenovirus, respiratory syncytial virus, human metapneumovirus); and to describe their self-medication practices. METHODS: A cross-sectional study was conducted in six pharmacies in Guatemala City. Study personnel collected nasopharyngeal and oropharyngeal swabs from participants who met the ILI case definition and who were self-medicating for the current episode. Participants were tested for influenza A and B and other pathogens using real-time RT-PCR. Participants' ILI-associated self-medication practices were documented using a questionnaire. RESULTS: Of all patients entering the pharmacy during peak hours who responded to a screening survey (n = 18,016) 6% (n = 1029) self-reported ILI symptoms, of which 45% (n = 470/1029) met the study case definition of ILI. Thirty-one percent (148/470) met inclusion criteria, of which 87% (130/148) accepted participation and were enrolled in the study. Among 130 participants, nearly half tested positive for viral infection (n = 55, 42.3%) and belonged to groups at low risk for complications from influenza. The prevalence of influenza A was 29% (n = 35). Thirteen percent of the study population (n = 17) tested positive for a respiratory virus other than influenza. Sixty-four percent of participants (n = 83) reported interest in receiving influenza vaccination if it were to become available in the pharmacy. Medications purchased included symptom-relieving multi-ingredient cold medications (n = 43/100, 43%), nonsteroidal anti-inflammatory drugs (n = 23, 23%), and antibiotics (n = 16, 16%). Antibiotic use was essentially equal among antibiotic users regardless of viral status. The broad-spectrum antibiotics ceftriaxone and azithromycin were the most common antibiotics purchased. CONCLUSIONS: During a typical influenza season, a relatively low proportion of all pharmacy visitors were experiencing influenza symptoms. A high proportion of clients presenting to pharmacies with ILI tested positive for a respiratory virus. Programs that guide appropriate use of antibiotics in this population are needed and become increasingly important during pandemics caused by respiratory viral pathogens.

A Candida albicans mannoprotein deprived of its mannan moiety is efficiently taken up and processed by human dendritic cells and induces T-cell activation without stimulating proinflammatory cytokine production.

Mannoproteins are cell wall components of pathogenic fungi and play major virulence and immunogenic roles with both their mannan and protein moieties. The 65-kDa mannoprotein (MP65) of Candida albicans is a beta-glucanase adhesin recognized as a major target of the human immune response against this fungus, and its recombinant product (rMP65; devoid of the mannan moiety) is presently under consideration as a vaccine candidate. Here we investigated cellular and molecular aspects of the interaction of rMP65 with human antigen-presenting cells. We also assessed the ability of rMP65 to initiate a T-cell response. Both the native mannosylated MP65 (nMP65) and the recombinant product were efficiently bound and taken up by macrophages and dendritic cells. However, contrarily to nMP65, rMP65 did not induce tumor necrosis factor alpha and interleukin-6 release from these cells. On the other hand, rMP65 was rapidly endocytosed by both macrophages and dendritic cells, in a process involving both clathrin-dependent and clathrin-independent mechanisms. Moreover, the RGD sequence inhibited rMP65 uptake to some extent. After internalization, rMP65 partially colocalized with lysosomal membrane-associated glycoproteins 1 and 2. This possibly resulted in efficient protein degradation and presentation to CD4(+) T cells, which proliferated and produced gamma interferon. Collectively, these results demonstrate that the absence of the mannan moiety does not deprive MP65 of the capacity to initiate the pattern of cellular and molecular events leading to antigen presentation and T-cell activation, which are essential features for further consideration of MP65 as a potential vaccine candidate.

An anti-beta-glucan monoclonal antibody inhibits growth and capsule formation of Cryptococcus neoformans in vitro and exerts therapeutic, anticryptococcal activity in vivo.

In this study we tested the in vitro and in vivo anti-Cryptococcus neoformans activity of an antilaminarin (anti-beta-glucan) monoclonal antibody (MAb 2G8) (immunoglobulin G2b) which was previously shown to inhibit the growth of beta-glucan-exposing Candida albicans cells. Here we show that MAb 2G8 binds to the cell wall of C. neoformans and inhibits its growth to an extent comparable to that observed for C. albicans. Binding and growth inhibition were detected almost equally for encapsulated and acapsular C. neoformans strains. In addition, at subinhibitory concentrations, MAb 2G8 reduced the capsule thickness without affecting protease or phospholipase production. Acapsular fungal cells, but not encapsulated fungal cells, were opsonized by the antibody and more efficiently phagocytosed and killed by human monocytes and by murine peritoneal macrophages. A single administration of MAb 2G8 resulted in a reduction in the fungal burden in the brains and livers of mice systemically infected with a highly virulent, encapsulated C. neoformans strain. This protective effect was also detected in neutropenic mice. Overall, these findings demonstrate that cell wall beta-glucan of encapsulated C. neoformans is accessible to antibodies which can exert remarkable anticryptococcal activities in vitro and in vivo.

An early imbalance of interleukin 12 influences the adjuvant effect of mannoproteins of Cryptococcus neoformans.

Mannoprotein from Cryptococcus neoformans induces protective response against a lethal challenge with this fungus or with Candida albicans. This phenomenon is largely related to early production of interleukin 12 (IL-12) and induction of T helper 1 response. Our study assesses whether the early absence of this critical cytokine could account for the incomplete activation of cellular response and whether the immune system compensates this imbalance. The results show that the neutralization of early IL-12 enhanced IL-18 production but decreased IFN-gamma secretion and IL-12R expression by splenic CD4 T cells. In contrast, IL-18R was not augmented despite an increase in IL-18 production. The co-stimulatory pathway was partially dysregulated because splenic macrophages showed unmodified B7-2, and a decrease of B7-1 expression. This dysregulation led to incomplete proliferative response of T cells in response to Cryptococcus neoformans and to increased fungal load in the brain 21 days post infection. The inability to dispose early IL-12, forced the immune system to compensate the imbalance and produced a series of long-lasting dysregulations involving the co-stimulatory pathway and T cell activation.

Disruption of CD40/CD40L interaction influences the course of Cryptococcus neoformans infection.

CD40 signaling has been implicated in various pathogenic processes such as chronic inflammatory disease, graft-versus-host disease, autoimmune disease and cancer. We previously demonstrated in an in vitro system that the CD40/CD40L pathway mediates late interleukin (IL) 12 production in response to Cryptococcus neoformans. The purpose of this study was to examine the course of C. neoformans infection in the absence of CD40/CD40L costimulation. We compared infection in mice genetically lacking CD40L (CD40L(-/-)) and in the wild-type counterpart. The animals were injected intratracheally with C. neoformans and monitored for clearance of the organism and the development of cellular immune response. CD40L(-/-) mice exhibited an exacerbation of infection, evaluated as scarce inflammatory response in the lung, that mirrored an increase of fungal burden. This correlated with impairment of nitrite production and antimicrobial activity by macrophages against C. neoformans and unrelated microorganisms such as Candida albicans. Moreover, IL-12 production by splenic macrophages was diminished in CD40L(-/-) mice and interferon-gamma production by CD4 and CD8 T cells was decreased. CD4 T cells retained the ability to express a costimulatory molecule, CTLA-4, but showed a decrease in CD28 expression. This latter molecule is implicated in a positive effect on proliferation, cytokine production and survival of T cells. Collectively these data demonstrate that absence of CD40L correlates with (i) reduced antimicrobial activity of natural effector cells; (ii) reduction of the magnitude of T cell response; and (iii) increase of fungal growth in the brain. These findings suggest that disruption of CD40/CD40L may be deleterious for development of an efficient immune response to C. neoformans and may identify potential molecular targets for novel immunotherapeutic approaches

The polysaccharide capsule of Cryptococcus neoformans interferes with human dendritic cell maturation and activation.

The ability of encapsulated and acapsular strains of Cryptococcus neoformans to activate dendritic cells (DC) derived from monocytes stimulated with granulocyte macrophage-colony stimulating factor and interleukin-4 was evaluated. Profound differences in DC response to encapsulated and acapsular C. neoformans strains were observed. In particular, (i) the acapsular strain was easily phagocytosed by immature DC, and the process induced several molecular markers, such as major histocompatibility complex (MHC) class I and class II, CD40, and CD83, which are characteristic of mature DC; (ii) the encapsulated strain did not up-regulate MHC class I and class II and CD83 molecules; (iii) the soluble capsular polysaccharide glucuronoxylomannan (GXM) is unable to regulate MHC class I and class II molecules; (iv) the addition of monoclonal antibody to GXM (anti-GXM) to the encapsulated strain facilitated antigen-presenting cell maturation by promoting ingestion of C. neoformans via Fc receptor for immunoglobulin G (FcgammaR)II (CD32) and FcgammaRIII (CD16); (v) pertubation of FcRgammaII or FcgammaRIII was insufficient to promote DC maturation; and (vi) optimal DC maturation permitted efficient T cell activation and differentiation, as documented by the enhancement of lymphoproliferation and interferon-gamma production. These results indicate that the C. neoformans capsule interferes with DC activation and maturation, indicating a new pathway by which the fungus may avoid an efficient T cell response.

Phenotypic switching of Cryptococcus neoformans can influence the outcome of the human immune response.

The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.

Mannoprotein from Cryptococcus neoformans promotes T-helper type 1 anticandidal responses in mice.

We previously demonstrated that mannoprotein (MP) from Cryptococcus neoformans (CnMP) stimulates interleukin-12 production by human monocytes, thus fostering a T-helper type 1 (Th1) protective anticryptococcal response. In this paper we show that CnMP was also able to induce a Candida albicans-directed protective Th1 response. This was demonstrated for mice immunized with CnMP by induction of a delayed-type hypersensitivity (DTH) reaction to C. albicans MP (CaMP) as well as induction of gamma interferon production by CD4(+) and CD8(+) splenic T cells stimulated in vitro with CaMP. CnMP-immunized mice were also partially protected from lethal systemic challenge with C. albicans, as shown by prolonged median survival times and decreased fungal burden in the kidney. Much evidence supports the validity of these cross-reactive and functional Th1 responses: (i) a non-cross-reactive C. albicans antigen, such as enolase, did not produce a DTH response to CaMP; (ii) passive adoptive transfer of T cells primed with CnMP induced a DTH reaction; (iii) C. neoformans extract elicited a DTH response to CaMP; and (iv) a monoclonal antibody (7H6) directed against a major and immunodominant T-cell-stimulatory 65-kDa MP (MP65) of C. albicans also recognized discrete 100-kDa constituents of C. neoformans extracts, as well as secretory constituents of the fungus. These results suggest the presence of common Th1 antigenic determinants in the mannoproteic material of C. neoformans and C. albicans epitopes, which should be considered in devising common strategies for immunoprophylactic or immunotherapeutic control of the fungi.