Recurrent visceral leishmaniasis relapses in HIV co-infected patients are characterized by less efficient immune responses and higher parasite load

Abstract

Visceral leishmaniasis (VL) and HIV co-infection (VL/HIV) has emerged as a significant public health problem in Ethiopia, with up to 30% of patients with VL co-infected with HIV. These patients suffer from recurrent VL relapses and increased mortality. Those with a previous history of VL relapses (recurrent VL/HIV) experience increased VL relapses as compared to patients with HIV presenting with their first episode of VL (primary VL/HIV). Our aim was to identify drivers that account for the higher rate of VL relapses in patients with recurrent VL/HIV (n = 28) as compared to primary VL/HIV (n = 21). Our results show that the relapse-free survival in patients with recurrent VL/HIV was shorter, that they had higher parasite load, lower weight gain, and lower recovery of all blood cell lineages. Their poorer prognosis was characterized by lower production of IFN-gamma, lower CD4⁺ T cell counts, and higher expression of programmed cell death protein 1 (PD1) on T cells.

Keywords: Immune response; Immunology; Virology.

Graphical abstract

Figure 1

Follow-up of patients with primary and recurrent VL/HIV 21 patients with P and 28 patients with R VL/HIV were followed for up to 6–12m. ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6–12m = 6–12 months post EoT.

Figure 2

Relapse-free survival Kaplan-Meier curves of participant VL relapses comparing patients with P VL/HIV to R VL/HIV. The hazard ratios (with 95% confidence intervals and p values) obtained from the Cox model indicated the change in relapse-free survival following treatment for VL for these groups. HR, hazard ration; CI, confidence interval.

Figure 3

Parasite load (A) Quantification of Leishmania amastigotes in smears of splenic aspirates collected from patients with P VL/HIV (n = 16) and R VL/HIV (n = 26) at ToD. (B) Quantification of the total expression of L. donovani (LD) mRNA in blood from patients with P (ToD: n = 12, EoT: n = 16, 3m: n = 8, 6-12m: n = 7) and R VL/HIV (ToD: n = 23, EoT: n = 14, 3m: n = 16, 6-12m: n = 18). (C) Comparison of the total expression of LD mRNA in blood from patients with P VL/HIV who did not relapse (n = 7) and those who did relapse (n = 1) and R VL/HIV who did not relapse (n = 6) and those who did relapse (n = 10) 3m after successful anti-leishmanial treatment. (D) Comparison of the total expression of L. donovani mRNA in blood from P VL/HIV who did not relapse (n = 5) and those who did relapse (n = 2) and R VL/HIV who did not relapse (n = 6) and those who did relapse (n = 12) 6–12m after successful anti-leishmanial treatment. If a patient did not relapse during the 2 time points of follow-up and if a patient relapsed at both 3, 6–12 months, this is represented as 2 measurements. ns = not significant. Each symbol represents the value for one individual, the straight lines represent the median. Statistical differences between P and R VL/HIV patients (A) at each time point (B) were determined using a Mann-Whitney test; statistical differences between the 4 different time points (B) for each cohort of patients were determined by Kruskal-Wallis test.LD mRNA = L. donovani mRNA. ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6-12m = 6–12 months post EoT. See also Table S1.

Figure 4

Clinical parameters (A) Body temperature was measured in patients with P VL/HIV (ToD: n = 21, EoT: n = 16, 3m: n = 14, 6-12m: n = 11) and R VL/HIV (ToD: n = 28, EoT: n = 22, 3m: n = 18, 6-12m: n = 13). (B) BMI was calculated for patients with P VL/HIV (ToD: n = 21, EoT: n = 17, 3m: n = 14, 6-12m: n = 12) and R VL/HIV (ToD: n = 28, EoT: n = 22, 3m: n = 18, 6-12m: n = 13). Each symbol represents the value for one individual, and the straight lines represent the median. Statistical differences between patients with P VL/HIV and R VL/HIV at each time point were determined using a Mann-Whitney test; statistical differences between the 4 different time points for each cohort of patients were determined by Kruskal-Wallis test. ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6-12m = 6–12 months post EoT. ns = not significant.

Figure 5

Spleen sizes (A) Spleen size as measured in cm below the costal margin on patients with P VL/HIV (ToD: n = 21, EoT: n = 17, 3m: n = 14, 6–12m: n = 11) and R VL/HIV (ToD: n = 28, EoT: n = 22, 3m: n = 18, 6–12m: n = 13). (B) Comparison of the spleen of patients with P VL/HIV who did not relapse (n = 12) and those who did relapse (n = 2) and patients with R VL/HIV who did not relapse (n = 7) and those who did relapse (n = 11) 3m after successful anti-leishmanial treatment. (C) Comparison of the spleen of patients with P VL/HIV who did not relapse (n = 9) and those who did relapse (n = 2) and patients with R VL/HIV who did not relapse (n = 5) and those who did relapse (n = 8) 6–12m after successful anti-leishmanial treatment. If a patient did not relapse during the 2 time points of follow-up and if a patient relapsed at both 3, 6–12 months, this is represented as 2 measurements. Each symbol represents the value for one individual, the straight lines represent the median. Statistical differences between 2 groups were determined using a Mann-Whitney test; statistical differences between the 4 different time points for each cohort of patients were determined by Kruskal-Wallis test.ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6-12m = 6–12 months post EoT. ns = not significant.

Figure 6

Whole blood assay: antigen-specific production of IFNγ and IL-10 Whole blood cells from patients with P VL/HIV (ToD: n = 16, EoT: n = 16, 3m: n = 9, 6-12m: n = 6) and R VL/HIV (ToD: n = 22, EoT: n = 23, 3m: n = 16, 6-12m: n = 17) were cultured in the presence of SLA and PBS. (A) IFNγ and (D). IL-10 levels in the supernatant were measured by ELISA after 24 h and the values obtained with PBS alone was deducted from the value obtained with SLA. (B) Comparison of IFNγ levels in the supernatant of whole blood cells from patients with P VL/HIV who did not relapse (n = 8) and those who did relapse (n = 1) and patients with R VL/HIV who did not relapse (n = 7) and those who did relapse (n = 9) 3m after successful anti-leishmanial treatment. (C) Comparison of IFNγ levels in the supernatant of whole blood cells from patients with P VL/HIV who did not relapse (n = 5) and those who did relapse (n = 1) and patients with R VL/HIV who did not relapse (n = 5) and those who did relapse (n = 12) 6-12m after successful anti-leishmanial treatment. If a patient did not relapse during the 2 time points of follow-up and if a patient relapsed at both 3, 6–12 months, this is represented as 2 measurements. Statistical differences between two groups were determined using a Mann-Whitney test; statistical differences between the 4 different time points for each cohort of patients were determined by Kruskal-Wallis test.ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6-12m = 6–12 months post EoT. ns = not significant. See also Table S5.

Figure 7

CD4⁺ and CD8⁺ T cell counts (A) CD4⁺ T cell counts in the blood of patients with P VL/HIV (ToD: n = 11, EoT: n = 10; 3m: n = 8, 6-12m: n = 9) and R VL/HIV (ToD: n = 17, EoT: n = 14, 3m: n = 13, 6-12m: n = 16). (B) Comparison of CD4⁺ T cell counts in the blood of patients with P VL/HIV who did not relapse (n = 7) and those who did relapse (n = 1) and patients with R VL/HIV who did not relapse (n = 6) and those who did relapse (n = 7) 3m after successful anti-leishmanial treatment. (C) Comparison of CD4⁺ T cell counts in the blood of patients with P VL/HIV who did not relapse (n = 8) and those who did relapse (n = 1) and patients with R VL/HIV who did not relapse (n = 5) and those who did relapse (n = 11) during 6-12m after successful anti-leishmanial treatment. (D) CD8⁺ T cell counts in the blood of patients with P VL/HIV (ToD: n = 13, EoT: n = 12, 3m: n = 8, 6-12m: n = 6) and R VL/HIV (ToD: n = 11, EoT: n = 12, 3m: n = 13, 6-12m: n = 10) were measured by flow cytometry. If a patient did not relapse during the 2 time points of follow-up and if a patient relapsed at both 3, 6–12 months, this is represented as 2 measurements. Statistical differences between two groups were determined using a Mann-Whitney test; statistical differences between the 4 different time points for each cohort of patients were determined by Kruskal-Wallis test.ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6-12m = 6–12 months post EoT. ns = not significant.

Figure 8

PD1 expression on CD4⁺ and CD8⁺ T cells (A) CD4 PD1 iMFI and (D). CD8 PD1 iMFI was measured by multiplying the % of T cells and the median fluorescence intensity of PD1 as measured by flow cytometry in the PBMCs of P VL/HIV (ToD: n = 13, EoT: n = 15, 3m: n = 10, 6-12m: n = 11), R VL/HIV patients (ToD: n = 15, EoT: n = 17, 3m: n = 16, 6-12m: n = 21) and healthy controls (n = 10). (B) Comparison of CD4 PD1 iMFI in patients with P VL/HIV who did not relapse (n = 9) and those who did relapse (n = 1) and patients with R VL/HIV who did not relapse (n = 9) and those who did relapse (n = 7) 3m after successful anti-leishmanial treatment. (C) Comparison of CD4 PD1 iMFI in patients with P VL/HIV who did not relapse (n = 8) and those who did relapse (n = 3) and patients with R VL/HIV who did not relapse (n = 7) and those who did relapse (n = 14) 6-12m after successful anti-leishmanial treatment. If a patient did not relapse during the 2 time points of follow-up and if a patient relapsed at both 3, 6–12 months, this is represented as 2 measurements. Statistical differences between two groups were determined using a Mann-Whitney test; statistical differences between the 4 different time points for each cohort of patients were determined by Kruskal-Wallis test.ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6-12m = 6–12 months post EoT. ns = not significant.