Evaluation of the 2007 WHO guideline to diagnose smear negative tuberculosis in an urban hospital in Ethiopia.

BACKGROUND: The 2007 World Health Organization (WHO) guideline to diagnose smear-negative tuberculosis (TB) in HIV-prevalent settings was mainly based on expert advice and therefore requires evaluation in real life situations. METHODS: In 2009, this guideline was introduced at the ALERT hospital in Ethiopia. From October 2009 to January 2011, the accuracy of the guideline was evaluated using Mycobacterium tuberculosis culture positivity as reference standard in HIV positive TB suspects. RESULTS: A total of 459 TB suspects were enrolled during the study period; 336 (73.2%) were HIV positive. Acid fast bacilli sputum smear microscopy was done for 74.7% (251/336) HIV positive TB suspects; 94.4% (237/251) were smear negative. A chest X-ray was performed in 92.8% (220/237) and a Mycobacterium tuberculosis culture in 63.7% (151/237). The median TB diagnostic delay for smear negative cases was 3 days (interquartile range 3-4 days). Of the 75 patients diagnosed with smear negative pulmonary TB, 89. 4% (67/75) were diagnosed by chest X-ray, 9.4% (7/75) by culture and 1.3% (1/75) by clinical suspicion only. In 147 smear negative TB suspects Mycobacterium tuberculosis culture and chest X-ray results were available. Among these 147 patients, the sensitivity of the chest X-ray to diagnose smear negative TB in HIV-positive TB suspects was 53.3% (95% CI: 26.7-78.7); the specificity 67.4% (95% CI: 58.7-75.3). CONCLUSION: The 2007 WHO diagnostic algorithm for the diagnosis of smear negative TB is likely to reduce the diagnostic delay and therefore decrease morbidity and mortality of TB in a HIV prevalent settings like Ethiopia

Whole Blood Stimulation Assay as a Treatment Outcome Monitoring Tool for VL Patients in Ethiopia: A Pilot Evaluation.

Visceral leishmaniasis (VL) is a lethal disease if left untreated. Current treatments produce variable rates of treatment failure and toxicity without sterile cure, rendering treatment efficacy monitoring essential. To avoid repeated invasive tissue aspirates as well as empirical treatment, there is a need for new tools that allow a less-invasive and early assessment of treatment efficacy in the field. Cross-sectional studies have suggested levels of cytokines/chemokines after whole blood stimulation as good markers of cure, but longitudinal studies are lacking. In this study, we followed 13 active VL cases in an endemic area in Ethiopia by measuring the production of IFN-γ, TNF-α, IP-10, IL-2, IL-10, MCP-1, and MIG before, during, and at the end of treatment. After 24 hours of stimulation of whole blood with soluble Leishmania antigen, we observed an early, robust, and incremental increase of IFN-γ, TNF-α, and IP-10 levels in all patients during treatment. Moreover, based on the IFN-γ levels that showed an average 13-fold increase from the time of diagnosis until the end of treatment, we could almost perfectly discriminate active from cured status. Similar concentrations and patterns were found in stimulation assays with the two main Leishmania species. The levels of IFN-γ, IP-10, or TNF-α also seemed to be inversely associated with the parasite load at baseline. Despite a 1/10 drop in concentrations, similar patterns were observed in IFN-γ and IP-10 levels when dried plasma spots were stored at 4°C for an average of 225 days. All the above evidence suggests a detectable restoration of cell-mediated immunity in VL and its association with parasite clearance. With a potential application in rural settings by means of dried plasma spots, we recommend to further explore the early diagnostic value of such assays for treatment efficacy monitoring in large cohort studies including treatment failure cases.Funding was provided by the Belgian Directorate-General for Development Cooperation under the ITM-DGDC framework agreement FA-IIII. WA is personally supported by a Research Foundation Flanders postdoctoral fellowship. In addition, this work was funded by the Instituto de Salud Carlos III via the project PI18CIII/00029 and via the Red de Enfermedades Tropicales, Subprograma RETICS del Plan Estatal de I+D+I 2013-2016, which is cofunded by FEDER “Una manera de hacer Europa” funds, via projects RD16/0027/0017 and RD16CIII/0003/0002.S

Dolutegravir based therapy showed CD4+ T cell count recovery and viral load suppression among ART naïve people living with HIV AIDS: a pilot evaluation

Abstract Recently, dolutegravir (DTG)-based combined therapy, a more effective and safer first-line antiretroviral therapy (ART), has been recommended by the World Health Organization for the treatment of Human Immunodeficiency Virus (HIV) since July 2018. However, its effectiveness in CD4+ T-cells count recovery and viral load suppression has not been studied yet in Ethiopia, where HIV is endemic. Therefore, we aimed to conduct a pilot assessment on the effect of DTG-based therapy on CD4+ T-cell count and viral load count among people living with HIV (PLWH) in Ethiopia. A longitudinal prospective cohort study was conducted from July 2020 to February 2021. 109 PLWH who are ART naive but plan to initiate DTG-based therapy were recruited. HIV viral ribonucleic acid (RNA) copies were determined using polymerase chain reaction. To compute the difference in viral load and CD4+ T-cell counts between the baseline, 3rd, and 6th months, a Friedman test was used. The study included 109 PLWH who had never received antiretroviral medication. Participants taking DTG-based treatment showed significantly decreasing median (IQR) values of viral load count (copies/mL) from 446,812 (237649.5–732994.5) at baseline to 34 (23.5–46) at 3 months and 0.0 (0–19) at 6 months of treatment follow-up. Although the treatment increases the proportion of participants with HIV-1 RNA 50 copies/mL from 0 (0% at baseline) to 87 (79.8%) and 100 (91.7%) at the 3rd and 6th months of treatment, respectively, On the other hand, the CD4+ T-cell count increased significantly during treatment: median (IQR): 209 (81.5–417.5) versus 291 (132–522) versus 378 (181–632.5) cells/L at baseline, the 3rd and 6th months of the treatment follow-up period, respectively. We found dolutegravir-based therapy was a promising option with high virological suppression rates and CD4+ T-cell count recovery, demonstrating a restoration of cellular immunity. Moreover, Viral load suppression rates were high after the initiation of the treatment. We recommend further research should be conducted with a larger number of participants to acquire greater awareness of the treatment outcomes

Helminth species specific expansion and increased TNF-alpha production of non-classical monocytes during active tuberculosis

Author summary Monocytes are important cells for the early innate immune response and play an integral part during inflammation and infection. Classical monocytes, the dominant monocyte subset during homeostasis and health, have been linked to efficient TB protection. Intermediate or non-classical monocytes have instead been associated with uncontrolled inflammation (TNF-alpha), cell death, and poor protection against Mycobacterium tuberculosis. In areas endemic for intestinal helminths, the immunoregulatory effects of monocytes may affect development or progression of TB disease. The role of monocyte subsets during helminth/TB coinfection have not been studied. In Gondar, Ethiopia, we show that in patients with helminth infection, a helminth species dependent expansion of non-classical monocytes is triggered, where Ascaris and hookworm had the strongest effect in coinfected pulmonary TB-patients. The increase in non-classical monocytes was mainly detected in coinfected patients with a low-to-intermediate disease severity. Only coinfection with helminths and TB induced an increased TNF-alpha response in monocytes. Thus, we found a helminth species-specific dysregulation of monocyte subset distribution and functionality in coinfected TB-patients which could affect TB pathogenesis. Both Mycobacterium tuberculosis infection and helminths may affect innate immune mechanisms such as differential effects on monocytes towards the non-classical and intermediate subsets that favor bacterial persistence. Our aim, was to investigate helminth species specific effects on the frequency and functional activity of monocyte subsets in patients with active tuberculosis and healthy subjects. HIV-negative patients with active pulmonary tuberculosis (PTB) and community controls (CCs) in Gondar, Ethiopia were screened for helminth infection by stool microscopy. Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and ex vivo stimulation with purified protein derivative (PPD) and helminth antigens were used to characterize the distribution of monocyte subsets and their function. A total of 74 PTB patients and 57 CCs with and without helminth infection were included. Non-classical monocytes were increased in PTB patients with Ascaris and hookworm infection but not in Schistosoma-infected patients. Ascaris had the strongest effect in increasing the frequency of non-classical monocytes in both PTB patients and CCs, whereas PTB without helminth infection did not affect the frequency of monocyte subsets. There was a helminth specific increase in the frequency of TNF-alpha producing non-classical monocytes in hookworm infected PTB patients, both with and without PPD-stimulation. Low-to-intermediate TB disease severity associated with increased frequency of non-classical monocytes only for helminth-positive PTB patients, and the frequency of TNF-alpha producing monocytes were significantly higher in intermediate and non-classical monocytes of helminth positive PTB patients with an intermediate disease score. Helminth infection affected the frequency of monocyte subsets and function both in TB patients and controls which was helminth species dependent in TB patients. The clinical role of this potential immunomodulatory effect needs further study and may affect the response and protection to tuberculosis in areas where helminth infections are endemic.Funding Agencies|Swedish Heart-Lung FoundationSwedish Heart-Lung Foundation [20160431, 20190484]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [201705617]</p

Helminth species dependent effects on Th1 and Th17 cytokines in active tuberculosis patients and healthy community controls

Despite that the impact of different helminth species is not well explored, the current dogma states that helminths affect the Th1/Th2 balance which in turn affects the risk of tuberculosis (TB) reactivation and severity of disease. We investigated the influence of helminth species on cytokine profiles including IL-17A in TB patients and healthy community controls (CCs). In total, 104 newly diagnosed pulmonary TB patients and 70 HIV negative and Quanti-FERON negative CCs in Gondar, Ethiopia were included following helminth screening by stool microscopy. Plasma samples and ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with purified protein derivative (PPD) and Staphylococcus enterotoxin B (SEB) was used to determine cytokine profiles by cytometric bead array. In CCs, Ascaris lumbricoides or Schistosoma mansoni infections were associated with an impaired Th1-type response (IFN-gamma, IL-6 and TNF-alpha) in PBMCs mainly with SEB stimulations, whereas in TB patients only hookworm infection showed a similar pattern. Among CCs, the IL-17A response in PBMCs stimulated with SEB was higher only for S. mansoni, whereas in TB patients, the elevated systemic IL-17A plasma level was significantly suppressed in hookworm infected TB patients compared to patients without helminth coinfection. Following treatment of TB and helminth infection there was a general decrease in ex vivio IL-10 and TNF-alpha production in unstimulated, PPD or SEB stimulated PBMCs that was the most pronounced and significant in TB patients infected with S. mansoni, whereas the follow-up levels of IFN-gamma and IL-17A was significantly increased only in TB patients without helminth coinfection from PBMCs stimulated mainly with SEB. In summary, in addition to confirming helminth specific effects on the Th1/Th2 response before and after TB treatment, our novel finding is that IL-17A was impaired in helminth infected TB patients especially for hookworm, indicating a helminth species-specific immunoregulatory effect on IL-17A which needs to be further investigated.Funding Agencies|Swedish Heart-Lung Foundation; Swedish Research Council</p

Helminth species-specific effects on IFN-gamma producing T cells during active and latent tuberculosis

BackgroundInterferon-gamma (IFN-gamma) is a key cytokine inducing protective immune responses during tuberculosis (TB) infection. Helminth-induced immune responses may affect IFN-gamma production by T cells, although its connection with disease severity and immune recovery during treatment is unexplored. We investigated the species-specific effect of helminths on the IFN-gamma production by T cells in relation to disease severity during active and latent TB infection (LTBI). MethodsIn this study, 69 active pulmonary TB patients (PTB), 28 with LTBI and 66 healthy controls were included. Active TB was diagnosed using GenXpert MTB/RIF while QuantiFERON test (QFT) was used for the screening of healthy community controls (CCs) and for the diagnosis of LTBI. Helminth infection was identified by routine diagnosis whereas clinical disease severity was evaluated by the TB score. Intracellular IFN-gamma production of T cells in stimulated peripheral blood mononuclear cells (PBMCs) was analyzed by flow cytometry using TB antigens (PPD), the polyclonal T cell activator staphylococcal enterotoxin B (SEB), or medium as unstimulated control. ResultsHelminth infected CCs and LTBI subjects showed a significant reduction of IFN-gamma(+) CD4(+) T cells by PPD-stimulation compared to non-helminth infected control groups. The significant reduction in the frequency of IFN-gamma(+) T cells in both latent and active PTB patients following SEB stimulation was mostly attributed to Schistosoma mansoni infection, whereas Ascaris lumbricoides, Schistosoma mansoni, and hookworm infection contributed equally in CCs. Following anti-helminthic and anti-TB treatment for 2 months, the frequency of IFN-gamma(+) CD4 T cells in helminth coinfected PTB was restored to levels of helminth negative PTB before treatment. Helminth coinfected PTB patients with an intermediate and severe clinical course had reduced capacity for production of IFN-gamma(+) T cells compared to the corresponding non-helminth infected PTB. ConclusionWe found a reduction in IFN-gamma producing T cells by helminth coinfection which was restored following anti-helminthic treatment. This reduction was helminth species-dependent in an exploratory sub-analysis and correlated to increased disease severity. Author summaryProtective immunity against tuberculosis (TB) requires a Th-1 response with cytokines like TNF and IFN-gamma which plays a key role in the recruitment and activation of immune cells. Helminth infection, on the other hand, can lead to induction of regulatory T cells and a Th-2 skewed response decreasing IFN-gamma in T cells. Decreased Th-1 responses could favor reactivation of latent TB infection (LTBI), although the helminth species-specific effect on IFN-gamma(+)CD4(+) T cells and the link to TB disease severity in patients with active pulmonary TB (PTB) have not been fully investigated. Therefore, blood cells (PBMCs) from healthy controls, LTBI individuals, and PTB patients were used to evaluate the impact of different helminths on the frequency of IFN-gamma(+)CD4(+) T cells, in Gondar Ethiopia. Ascaris lumbricoides, Schistosoma mansoni, and hookworm infection in healthy controls contributed equally to decreasing the frequency of IFN-gamma(+)CD4(+) T cells, whereas in both LTBI and PTB patients S. mansoni coinfection had the greatest impact on reducing IFN-gamma producing capacity of T cells. Decreased IFN-gamma producing capacity of T cells was correlated with increased TB disease severity, only in helminth coinfected PTB patients, and anti-helminthic therapy restored the IFN-gamma producing capacity of T cells at the 2 months follow-up.Funding Agencies|Swedish Heart-Lung Foundation (Hjart-Lungfonden); Swedish Research Council (Vetenskapsradet)</p

Successful Treatment of Human Visceral Leishmaniasis Restores Antigen-Specific IFN- γ, but not IL-10 Production

Abstract One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation

Successful treatment of human visceral leishmaniasis restores antigen-specific IFN-γ, but not IL-10 production

One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation