Tuberculosis in HIV-Negative and HIV-Infected Patients in a Low-Incidence Country: Clinical Characteristics and Treatment Outcomes

Background In Switzerland and other developed countries, the number of tuberculosis (TB) cases has been decreasing for decades, but HIV-infected patients and migrants remain risk groups. The aim of this study was to compare characteristics of TB in HIV-negative and HIV-infected patients diagnosed in Switzerland, and between coinfected patients enrolled and not enrolled in the national Swiss HIV Cohort Study (SHCS). Methods and Findings All patients diagnosed with culture-confirmed TB in the SHCS and a random sample of culture-confirmed cases reported to the national TB registry 2000–2008 were included. Outcomes were assessed in HIV-infected patients and considered successful in case of cure or treatment completion. Ninety-three SHCS patients and 288 patients selected randomly from 4221 registered patients were analyzed. The registry sample included 10 (3.5%) coinfected patients not enrolled in the SHCS: the estimated number of HIV-infected patients not enrolled in the SHCS but reported to the registry 2000–2008 was 146 (95% CI 122–173). Coinfected patients were more likely to be from sub-Saharan Africa (51.5% versus 15.8%, P<0.0001) and to present disseminated disease (23.9% vs. 3.4%, P<0.0001) than HIV-negative patients. Coinfected patients not enrolled in the SHCS were asylum seekers or migrant workers, with lower CD4 cell counts at TB diagnosis (median CD4 count 79 cells/µL compared to 149 cells/µL among SHCS patients, P = 0.07). There were 6 patients (60.0%) with successful outcomes compared to 82 (88.2%) patients in the SHCS (P = 0.023). Conclusions The clinical presentation of coinfected patients differed from HIV-negative TB patients. The number of HIV-infected patients diagnosed with TB outside the SHCS is similar to the number diagnosed within the cohort but outcomes are poorer in patients not followed up in the national cohort. Special efforts are required to address the needs of this vulnerable population.

Estimating loss to follow-up in HIV-infected patients on antiretroviral therapy: the effect of the competing risk of death in Zambia and Switzerland

Background Loss to follow-up (LTFU) is common in antiretroviral therapy (ART) programmes. Mortality is a competing risk (CR) for LTFU; however, it is often overlooked in cohort analyses. We examined how the CR of death affected LTFU estimates in Zambia and Switzerland. Methods and Findings HIV-infected patients aged ≥18 years who started ART 2004–2008 in observational cohorts in Zambia and Switzerland were included. We compared standard Kaplan-Meier curves with CR cumulative incidence. We calculated hazard ratios for LTFU across CD4 cell count strata using cause-specific Cox models, or Fine and Gray subdistribution models, adjusting for age, gender, body mass index and clinical stage. 89,339 patients from Zambia and 1,860 patients from Switzerland were included. 12,237 patients (13.7%) in Zambia and 129 patients (6.9%) in Switzerland were LTFU and 8,498 (9.5%) and 29 patients (1.6%), respectively, died. In Zambia, the probability of LTFU was overestimated in Kaplan-Meier curves: estimates at 3.5 years were 29.3% for patients starting ART with CD4 cells <100 cells/µl and 15.4% among patients starting with ≥350 cells/µL. The estimates from CR cumulative incidence were 22.9% and 13.6%, respectively. Little difference was found between naïve and CR analyses in Switzerland since only few patients died. The results from Cox and Fine and Gray models were similar: in Zambia the risk of loss to follow-up and death increased with decreasing CD4 counts at the start of ART, whereas in Switzerland there was a trend in the opposite direction, with patients with higher CD4 cell counts more likely to be lost to follow-up. Conclusions In ART programmes in low-income settings the competing risk of death can substantially bias standard analyses of LTFU. The CD4 cell count and other prognostic factors may be differentially associated with LTFU in low-income and high-income settings.

Prevalence and predictors of kaposi sarcoma herpes virus seropositivity: a cross-sectional analysis of HIV-infected adults initiating ART in Johannesburg, South Africa

Background Kaposi sarcoma (KS) is the most common AIDS-defining tumour in HIV-infected individuals in Africa. Kaposi sarcoma herpes virus (KSHV) infection precedes development of KS. KSHV co-infection may be associated with worse outcomes in HIV disease and elevated KSHV viral load may be an early marker for advanced HIV disease among untreated patients. We examined the prevalence of KSHV among adults initiating antiretroviral therapy (ART) and compared immunological, demographic and clinical factors between patients seropositive and seronegative for KSHV. Results We analyzed cross-sectional data collected from 404 HIV-infected treatment-naïve adults initiating ART at the Themba Lethu Clinic, Johannesburg, South Africa between November 2008 and March 2009. Subjects were screened at ART initiation for antibodies to KSHV lytic K8.1 and latent Orf73 antigens. Seropositivity to KSHV was defined as positive to either lytic KSHV K8.1 or latent KSHV Orf73 antibodies. KSHV viremia was determined by quantitative PCR and CD3, 4 and 8 lymphocyte counts were determined with flow cytometry. Of the 404 participants, 193 (48%) tested positive for KSHV at ART initiation; with 76 (39%) reactive to lytic K8.1, 35 (18%) to latent Orf73 and 82 (42%) to both. One individual presented with clinical KS at ART initiation. The KSHV infected group was similar to those without KSHV in terms of age, race, gender, ethnicity, smoking and alcohol use. KSHV infected individuals presented with slightly higher median CD3 (817 vs. 726 cells/mm3) and CD4 (90 vs. 80 cells/mm3) counts than KSHV negative subjects. We found no associations between KSHV seropositivity and body mass index, tuberculosis status, WHO stage, HIV RNA levels, full blood count or liver function tests at initiation. Those with detectable KSHV viremia (n = 19), however, appeared to present with signs of more advanced HIV disease including anemia and WHO stage 3 or 4 defining conditions compared to those in whom the virus was undetectable. Conclusions We demonstrate a high prevalence of KSHV among HIV-infected adults initiating ART in a large urban public-sector HIV clinic. KSHV viremia but not KSHV seropositivity may be associated with markers of advanced HIV disease.

In vitro induction of lymph node cell proliferation by mouse bone marrow dendritic cells following stimulation with different Echinococcus multilocularis antigens

The immune response of mice experimentally infected with Echinococcus multilocularis metacestodes becomes impaired so as to allow parasite survival and proliferation. Our study tackled the question on how different classes of E. multilocularis antigens (crude vesicular fluid (VF); purified proteinic rec-14-3-3; purified carbohydrate Em2(G11)) are involved in the maturation process of bone-marrow-derived dendritic cells (BMDCs) and subsequent exposure to lymph node (LN) cells. In our experiments, we used BMDCs cultivated from either naïve (control) or alveolar echinococcosis (AE)-infected C57BL/6 mice. We then tested surface markers (CD80, CD86, MHC class II) and cytokine expression levels (interleukin (IL)-10, IL-12p40 and tumour necrosis factor (TNF)-α) of non-stimulated BMDCs versus BMDCs stimulated with different Em-antigens or lipopolysaccharide (LPS). While LPS and rec-14-3-3-antigen were able to induce CD80, CD86 and (to a lower extent) MHC class II surface expression, Em2(G11) and, strikingly, also VF-antigen failed to do so. Similarly, LPS and rec-14-3-3 yielded elevated IL-12, TNF-α and IL-10 expression levels, while Em2(G11) and VF-antigen didn't. When naïve BMDCs were loaded with VF-antigen, they induced a strong non-specific proliferation of uncommitted LN cells. For both, BMDCs or LN cells, isolated from AE-infected mice, proliferation was abrogated. The most striking difference, revealed by comparing naïve with AE-BMDCs, was the complete inability of LPS-stimulated AE-BMDCs to activate lymphocytes from any LN cell group. Overall, the presenting activity of BMDCs from AE-infected mice seemed to trigger unresponsiveness in T cells, especially in the case of VF-antigen stimulation, thus contributing to the suppression of clonal expansion during the chronic phase of AE infection.

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

In vitro effects of arylimidamides against Besnoitia besnoiti infection in Vero cells

The in vitro effects of 4 arylimidamides (DB811, DB786, DB750 and DB766) against the proliferative tachyzoite stage of the apicomplexan parasite Besnoitia besnoiti were investigated. These four compounds had been shown earlier to exhibit in vitro activities in the nanomolar range against the related apicomplexans Neospora caninum and Toxoplasma gondii. Real-time-PCR was used to assess B. besnoiti intracellular proliferation in vitro. Preliminary assessment by light microscopy identified DB811 and DB750 as the most promising compounds, while DB786 and DB766 were much less effective. Three-day-growth assays and quantitative real-time PCR was used for IC50 determination of DB811 (0.079 muM) and DB750 (0.56 muM). Complete growth inhibition was observed at 1.6 muM for DB 811 and 1.7 muM for DB750. However, when infected cultures were treated for 14 days, proliferation of parasites occurred again in cultures treated with DB750 from day 4 onwards, while the proliferation of DB811-treated tachyzoites remained inhibited. Electron microscopy of B. besnoiti-infected fibroblast cultures fixed and processed at different time-points following the initiation of drug treatments revealed that DB811 exerted a much higher degree of ultrastructural alterations compared to DB750. These results show that arylimidamides such as DB811 could potentially become an important addition to the anti-parasitic arsenal for food animal production, especially in cattle.

Clinical, laboratory and pathological findings in dogs experimentally infected with Angiostrongylus vasorum

The aim of this comparative study was to investigate the development of clinical signs and accompanying haematological, coproscopic and pathological findings as a basis for the monitoring of health condition of Angiostrongylus vasorum infected dogs. Six beagles were orally inoculated with 50 (n=3) or 500 (n=3) A. vasorum third stage larvae (L3) obtained from experimentally infected Biomphalaria glabrata snails. Two dogs were treated with moxidectin/imidacloprid spot-on solution and two further dogs with an oral experimental compound 92 days post infection (dpi), and were necropsied 166 dpi. Two untreated control dogs were necropsied 97 dpi. Prepatency was 47-49 days. Dogs inoculated with 500 L3 exhibited earlier (from 42 dpi) and more severe respiratory signs. Clinical signs resolved 12 days after treatment and larval excretion stopped within 20 days in all four treated dogs. Upon necropsy, 10 and 170 adult worms were recovered from the untreated dogs inoculated with 50 and 500 L3, respectively. Adult worms were also found in two treated dogs, in the absence of L1 or eggs. Despite heavy A. vasorum infection load and severe pulmonary changes including vascular thrombosis, only mild haematological changes were observed. Eosinophilia was absent but the presence of plasma cells was observed. Neutrophilic leucocytes showed a transient increase but only after treatment. Signs for coagulopathies were slight; nevertheless coagulation parameters were inoculation dose dependent. Ten weeks after treatment pulmonary fibrosis was still present. Infections starting from 50 L3 of A. vasorum had a massive impact on lung tissues and therefore on the health of affected dogs, particularly after prepatency, although only mild haematological abnormalities were evident.

Low levels of mannan-binding lectin or ficolins are not associated with an increased risk of cytomegalovirus disease in HIV-infected patients

Background In HIV-infected patients, prediction of Cytomegalovirus (CMV) disease remains difficult. A protective role of mannan-binding lectin (MBL) and ficolins against CMV disease has been reported after transplantation, but the impact in HIV-infected patients is unclear. Methods In a case-control study nested within the Swiss HIV Cohort Study, we investigated associations between plasma levels of MBL/ficolins and CMV disease. We compared HIV-infected patients with CMV disease (cases) to CMV-seropositive patients without CMV disease (controls) matched for CD4 T-cells, sampling time, and use of combination antiretroviral therapy. MBL and M-ficolin, L-ficolin, and H-ficolin were quantified using ELISA. Results We analysed 105 cases and 105 matched controls. CMV disease was neither associated with MBL (odds ratio [OR] 1.03 per log10 ng/mL increase (95% CI 0.73–1.45)) nor with ficolins (OR per log10 ng/mL increase 0.66 (95% CI 0.28–1.52), 2.34 (95% CI 0.44–12.36), and 0.89 (95% CI 0.26–3.03) for M-ficolin, L-ficolin, and H-ficolin, respectively). We found no evidence of a greater association between MBL and CMV disease in patients with low CD4 counts; however in the multivariable analysis, CMV disease was more likely in patients with an increased HIV RNA (OR 1.53 per log10 copies/mL; 95% CI 1.08–2.16), or a shorter duration of HIV-infection (OR 0.91 per year; 95% CI 0.84–0.98). Conclusions CMV disease is not associated with low levels of MBL/ficolins, suggesting a lack of a protective role in HIV-infected patients.

Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection

HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.

CD4 cell count and the risk of AIDS or death in HIV-Infected adults on combination antiretroviral therapy with a suppressed viral load: a longitudinal cohort study from COHERE

Background Most adults infected with HIV achieve viral suppression within a year of starting combination antiretroviral therapy (cART). It is important to understand the risk of AIDS events or death for patients with a suppressed viral load. Methods and Findings Using data from the Collaboration of Observational HIV Epidemiological Research Europe (2010 merger), we assessed the risk of a new AIDS-defining event or death in successfully treated patients. We accumulated episodes of viral suppression for each patient while on cART, each episode beginning with the second of two consecutive plasma viral load measurements <50 copies/µl and ending with either a measurement >500 copies/µl, the first of two consecutive measurements between 50–500 copies/µl, cART interruption or administrative censoring. We used stratified multivariate Cox models to estimate the association between time updated CD4 cell count and a new AIDS event or death or death alone. 75,336 patients contributed 104,265 suppression episodes and were suppressed while on cART for a median 2.7 years. The mortality rate was 4.8 per 1,000 years of viral suppression. A higher CD4 cell count was always associated with a reduced risk of a new AIDS event or death; with a hazard ratio per 100 cells/µl (95% CI) of: 0.35 (0.30–0.40) for counts <200 cells/µl, 0.81 (0.71–0.92) for counts 200 to <350 cells/µl, 0.74 (0.66–0.83) for counts 350 to <500 cells/µl, and 0.96 (0.92–0.99) for counts ≥500 cells/µl. A higher CD4 cell count became even more beneficial over time for patients with CD4 cell counts <200 cells/µl. Conclusions Despite the low mortality rate, the risk of a new AIDS event or death follows a CD4 cell count gradient in patients with viral suppression. A higher CD4 cell count was associated with the greatest benefit for patients with a CD4 cell count <200 cells/µl but still some slight benefit for those with a CD4 cell count ≥500 cells/µl.

Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood-cerebrospinal fluid barrier

Enterovirus is the most common pathogen causing viral meningitis especially in children. Besides the blood-brain barrier (BBB) the choroid plexus, which forms the blood-cerebrospinal-fluid (CSF) barrier (BCSFB), was shown to be involved in the pathogenesis of enteroviral meningitis. In a human in vitro model of the BCSFB consisting of human choroid plexus papilloma cells (HIBCPP), the permissiveness of plexus epithelial cells for Echovirus 30 (EV30) was analyzed by immunoblotting and quantitative real-time PCR (Q-PCR). HIBCPP could be directly infected by EV30 from the apical as well as from the physiological relevant basolateral side. During an infection period of 5h no alterations of barrier function and cell viability could be observed. Analysis of the cytokine/chemokine-profile following enteroviral infection with a cytometric bead array (CBA) and Q-PCR revealed an enhanced secretion of PanGRO (CXCL1, CXCL2 and CXCL3), IL8 and CCL5. Q-PCR showed a significant upregulation of CXCL1, CXCL2 and CXCL3 in a time dependant manner. However, there was only a minor effect of HIBCPP-infection with EV30 on transepithelial T lymphocyte migration with or without the chemoattractant CXCL12. Moreover, CXCL3 did not significantly enhance T cell migrations. Therefore additional factors must be involved for the in vivo reported enhanced T cell migration into the CNS in the context of enteroviral meningitis. As HIBCPP are permissive for infection with EV30, they constitute a valuable human in vitro model to study viral infection at the BCSFB.

Determinants of hepatitis A vaccine immunity in a cohort of human immunodeficiency virus-infected children living in Switzerland

Vaccination in HIV-infected children is often less effective than in healthy children. The goal of this study was to assess vaccine responses to hepatitis A virus (HAV) in HIV-infected children. Children of the Swiss Mother and Child HIV Cohort Study (MoCHiV) were enrolled prospectively. Recommendations for initial, catch-up, and additional HAV immunizations were based upon baseline antibody concentrations and vaccine history. HAV IgG was assessed by enzyme-linked immunosorbent assay (ELISA) with a protective cutoff value defined as ≥10 mIU/ml. Eighty-seven patients were included (median age, 11 years; range, 3.4 to 21.2 years). Forty-two patients were seropositive (48.3%) for HAV. Among 45 (51.7%) seronegative patients, 36 had not received any HAV vaccine dose and were considered naïve. Vaccine responses were assessed after the first dose in 29/35 naïve patients and after the second dose in 33/39 children (25 initially naïve patients, 4 seronegative patients, and 4 seropositive patients that had already received 1 dose of vaccine). Seroconversion was 86% after 1 dose and 97% after 2 doses, with a geometric mean concentration of 962 mIU/ml after the second dose. A baseline CD4(+) T cell count below 750 cells/μl significantly reduced the post-2nd-dose response (P = 0.005). Despite a high rate of seroconversion, patients with CD4(+) T cell counts of <750/μl had lower anti-HAV antibody concentrations. This may translate into a shorter protection time. Hence, monitoring humoral immunity may be necessary to provide supplementary doses as needed.

Reduced nuclear translocation of nuclear factor (NF)-kappaB p65 in the footpad epidermis of dogs infected with distemper virus

Infection of canine footpads with the canine distemper virus (CDV) can cause massive epidermal thickening (hard pad disease), as a consequence of increased proliferation of keratinocytes and hyperkeratosis. Keratinocytes of canine footpad epidermis containing detectable CDV nucleoprotein antigen and CDV mRNA were shown previously to have increased proliferation indices. Because various proteins that play a role in the proliferation of epidermal cells are viral targets, the potential participation of such proteins in CDV-associated keratinocyte proliferation was investigated. Transforming growth factor-alpha (TGF-alpha), cell cycle regulatory proteins p21, p27 and p53, and nuclear factor (NF)-kappaB transcription factor components p50 and p65 were studied in the footpad epidermis from the following groups of dogs inoculated with CDV: group 1, consisting of seven dogs with clinical distemper and CDV in the footpad epidermis; group 2, consisting of four dogs with clinical distemper but no CDV in the footpad epidermis; group 3, consisting of eight dogs with neither clinical distemper nor CDV in the footpad epithelium. Group 4 consisted of two uninoculated control dogs. The expression of TGF-alpha, p21, p27 and p53, and p50 in the basal layer, lower and upper spinous layers, and in the granular layer did not differ statistically between CDV-positive (group 1) and CDV-negative (groups 2-4) footpad epidermis. However, there were differences in the levels of nuclear and cytoplasmic p65 expression between group 1 dogs and the other three groups. Thus, footpads from group 1 dogs had more keratinocytes containing p65 in the cytoplasm and, conversely, fewer nuclei that were positive for p65. These findings indicate that p65 translocation into the nucleus is reduced in CDV-infected footpad epidermis. Such decreased translocation of p65 may help to explain increased keratinocyte proliferation in hard pad disease and suggests interference of CDV with the NF-kappaB pathway.

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Bovine viral diarrhea virus (BVDV), together with Classical swine fever virus (CSFV) and Border disease virus (BDV) of sheep, belongs to the genus Pestivirus of the Flaviviridae. BVDV is either cytopathic (cp) or noncytopathic (ncp), as defined by its effect on cultured cells. Infection of pregnant animals with the ncp biotype may lead to the birth of persistently infected calves that are immunotolerant to the infecting viral strain. In addition to evading the adaptive immune system, BVDV evades key mechanisms of innate immunity. Previously, we showed that ncp BVDV inhibits the induction of apoptosis and alpha/beta interferon (IFN-alpha/beta) synthesis by double-stranded RNA (dsRNA). Here, we report that (i) both ncp and cp BVDV block the induction by dsRNA of the Mx protein (which can also be induced in the absence of IFN signaling); (ii) neither biotype blocks the activity of IFN; and (iii) once infection is established, BVDV is largely resistant to the activity of IFN-alpha/beta but (iv) does not interfere with the establishment of an antiviral state induced by IFN-alpha/beta against unrelated viruses. The results of our study suggest that, in persistent infection, BVDV is able to evade a central element of innate immunity directed against itself without generally compromising its activity against unrelated viruses ("nonself") that may replicate in cells infected with ncp BVDV. This highly selective "self" and "nonself" model of evasion of the interferon defense system may be a key element in the success of persistent infection in addition to immunotolerance initiated by the early time point of fetal infection.

Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.