Patients’ characteristics
Between the 1st of September 2020 and the 30th of April 2021, 126 patients living with IMID were included in the study. The median age was 44 ± 14 years, patients were 61% male and 39% female. Of these, 74% were diagnosed with intestinal inflammatory disease, while 24% presented with rheumatological diseases and 2% with both types of disorders (Table 1). One hundred-thirteen (90%) out of 126 patients were under treatment with TNF-α inhibitors, while the remaining patients were treated with other biologics including Abatacept, Vedolizumab, Baricitinib and/or Tocilizumab (Table 1).
Blood samples were analyzed for Leishmania spp. infection using three different techniques, and were considered positive if at least one of the methods yielded a positive result. Consequently, the control group was defined as those patients who tested negative in all three assays.
Results of molecular, serological and immunological tests
Real-time PCR targeting Leishmania kDNA tested positive in 3 out of 126 patients (2%). In particular, one of these patients had high parasitemia levels (9 × 105 equivalent parasites/mL) and exhibited suggestive symptoms of visceral leishmaniasis (VL), and was therefore diagnosed with overt disease. The two other PCR-positive patients had low parasite loads (6 × 10–1 and 2 × 10–1 equivalent parasites/mL, respectively) and were asymptomatic.
The presence of specific IgG antibody in serum was assessed using the WB, with 12% (15/126) of patients testing positive.
WBA test was conducted in 125 out of the 126 patients. Nine patients (7.2%) tested positive to at least one of the WBA targets. Of these, 4 patients tested positive for IP-10 alone, 3 patients were positive for both IL-2 and IP-10, while 2 patients were positive for IL-2 alone. Patients with positive WBA showed significantly higher values for IL-2 (median 10.85 pg/mL; p < 0.001) and IP-10 (median 533.0 pg/mL; p < 0.010) when compared to WBA-negative individuals (median 0.1 pg/mL and 5 pg/mL respectively) (Fig. 1).
IL-2 and IP-10 levels in soluble leishmania antigen (SLA)-stimulated whole blood from patients with immune-mediated inflammatory diseases residing in a L. infantum endemic area. POS, positive; NEG, negative. Horizontal bars represent median concentrations. Each dot represents one subject. ****p < 0.0001 by using Mann–Whitney U test
Among the 125 patients enrolled in the study who showed no symptoms of leishmaniasis, 25 were positive on at least one test, indicating a 20% prevalence of asymptomatic Leishmania infection in IMID. A significant difference emerged (p = 0.012) between the frequency of Leishmania-positive patients residing in northern Italy (17%, 18/109) and those residing in central and southern Italy (46%, 6/13). Concerning patients affected by intestinal IMID, 14% (7/51) of patients with Crohn’s disease had asymptomatic Leishmania infection, compared with 17% (7/42) of patients with rectal ulcerative colitis or ulcerative pancolitis, showing no significant differences between baseline inflammatory bowel disease and the presence of Leishmania spp. infection.
Among patients with arthritis, differences were noticed in terms of frequency of positivity to Leishmania infection in patients with rheumatoid arthritis (1/9, 11%), seronegative arthritis (2/3, 67%), psoriatic arthritis (3/3, 100%), and spondylarthritis (5/15, 33%); however, the sample size was too small in each subgroup of patients to draw any conclusion. In addition, we did not detect any difference due to sex or age, among individuals who tested positive in any Leishmania-specific test and Leishmania-negative individuals.
Concordance between tests in detecting Leishmania infection
Figure 2 presents a Venn diagram illustrating the overlap among PCR, WBA, and WB results in 26 patients who tested positive for at least one of the three methods; the VL case exhibited high parasitemia, tested positive by WB, but negative by WBA. Conversely, among patients with asymptomatic Leishmania infection (n = 25), none tested positive on at least two assays simultaneously. Contingency tables were used to assess concordance between the tests; each test showed poor or no agreement with the others (Cohen’s kappa coefficients were − 0.278 for PCR/WBA, 0.293 for WB/WBA, and 0.074 for PCR/WB).
Venn diagram showing the agreement among PCR (Polymerase chain reaction), WBA (Whole blood assay) and WB (Western blot) in Leishmania positive patients (n = 26). The overlapping regions represent the concordance of positive results in two or more tests
Diagnostic accuracy of serological, molecular and immunological tests
When assessing the sensitivity of each test or test combination with respect to the definition of asymptomatic Leishmania infection (n = 25), we observed that WB exhibited the highest sensitivity as a single test (14 positive patients, 56%), while WBA had a sensitivity of 36% (9 positive patients) and PCR exhibited a sensitivity of 8% (2 positive patients, Table 2). The combination of WB and WBA provided the best performance, with a sensitivity of 92% (23 positive patients out of 25) for detecting asymptomatically infected individuals.