This study showed a frequency of 15% of Leishmania spp. infection in PLWH, with weak concordance of the tests utilized. There are few studies evaluating the prevalence of Leishmania asymptomatic infection among PLWH, and there are no studies analysing this frequency in an urban area with no reports of autochthonous transmission of VL in Brazil. Taking into account the overall prevalence (at least one positive test) observed in this study, the result was less than that reported by Orsini et al., 20.2% [12]. In another study performed in Brazil, Carranza-Tamayo et al. showed similar data, with an overall prevalence of 16.0% [11]. Both studies were conducted in another area of the country (Federal District) and did not include data from Sao Paulo. There are no studies including patients from the Southeast Region.
Considering studies in other parts of the world, the frequency of coinfection is lower than that found by Garrote et al., 64.0%, in Spain [28]. These discrepancies are probably explained by the use of different diagnostic tests, including different antigens and molecular targets. Recently, in a systematic review of asymptomatic visceral leishmaniasis in the Indian subcontinent, Hirve et al. [29], including 31 articles with different tests (rK39 immunochromatographic test, rK39 ELISA, DAT, PCR or Leishmanin skin test), found a prevalence from 0.25% to 36.9%, depending on the method used. Recently, Echchakery et al. [18], in Morocco, found a prevalence of 5% in PLWH by indirect immunofluorescence. Clearly, there is no consensus about the best diagnostic tool to estimate the frequency of asymptomatic Leishmania infection in the population, and in the context of HIV infection, even in fully developed symptomatic disease, there are few data from Latin America regarding the performance of diagnostic methods [30].
The data also demonstrated poor agreement between serological tests. This fact has been shown by other prevalence studies involving PLWH and immunocompetent individuals [11, 12, 31, 32] and can be accounted for by the use of different antigens and molecular targets (surface, soluble or recombinant antigens) that can detect many stages of asymptomatic infection.
ELISA using Leptomonas seymouri antigen was performed for the first time in people with HIV infection in this study. It has already been demonstrated that this antigen has good performance for the diagnosis of visceral leishmaniasis; in fact, its performance is comparable to that of the L. chagasi antigen. Similar to other crude antigens, there is the possibility of cross-reactivity with T. cruzi and other Leishmania species [22]. Recently, Kesper et al. [33], while performing ELISA using Leptomonas seymouri and Crithidia fasciculata antigens, showed 100% reactivity with sera from visceral leishmaniasis (VL) cases and no reactivity with American tegumentary leishmaniasis (ATL). Nevertheless, these individuals did not have HIV infection. In this study, one patient had Chagas disease (undetermined form), and his serum was positive for Leptomonas antigen. We strongly suggest that further studies are necessary to determine the sensitivity and specificity for VL diagnosis in individuals with HIV infection using the Leptomonas antigen.
IFAT using L. major-like antigen is a test recommended by the Ministry of Health of Brazil for the diagnosis of leishmaniasis [34]. In this study, IFAT showed greater positivity compared to Leptomonas ELISA. In other studies evaluating the prevalence of Leishmania/HIV coinfection, ELISA using crude antigens demonstrated greater positivity compared to IFAT [11, 12], and this was attributed to the fact that IFAT detects mainly antibodies against surface antigens, while crude-antigen ELISA detects a wider variety of antibodies directed against soluble antigen components. In our study, this difference was not observed (Table 2) and was probably related to divergence between the specificity of these tests and the immunological characteristics of antigens.
Considering only serological methods, we found that a lower CD4+ T cell count was associated with a positive ELISA for Leptomonas or IFAT. These data can be, at first, considered surprising, given that serological methods have worse sensitivity for visceral leishmaniasis diagnosis in PLWH compared to immunocompetent individuals [8, 30, 35]. This concept is applicable to other infectious diseases, especially to the advanced stage of AIDS, in which severe T and B lymphocyte dysfunction triggers decreased production of specific antibodies [5]. It is important to highlight that all this information is related to the performance of diagnosis tests in subjects with symptomatic visceral leishmaniasis, and herein, we discuss asymptomatic individuals. Another important point is the possibility of cross-reaction of these tests. Although there was no association between current disease in treatment and test positivity, it is possible that these individuals with lower values of CD4+ T cell counts had subclinical or latent diseases, which may have interfered with the results. Another question is the possible variability of humoral response in PLWH. Gradoni et al [36], comparing serological IFAT results of HIV-infected and non-HIV-infected individuals with visceral leishmaniasis, showed worse sensitivity of this method in those infected with HIV and a greater variability of results in this group, with some people with IgG levels far above the non-HIV-infected subjects. These findings may reflect the temporal sequence of acquisition of the two infectious agents (HIV and Leishmania). Individuals who acquire Leishmania infection before HIV infection should produce high levels of IgG directed against antigens recognized before T cell impairment due to HIV. This response is probably related to increased IgG production because of nonspecific polyclonal B cell activation, which occurs more frequently in people with severe immunosuppression [5, 36]. This abnormal humoral response, more evident in severely immunosuppressed patients, can explain a greater proportion of positive serological tests in the group, considering that all samples are from individuals currently living in a non-endemic area for VL that probably acquired Leishmania infection before HIV infection.
rK39 ELISA had the lowest positivity among all serological methods. This antigen has been used both in ELISA and in rapid diagnostic tests [37-39], including in PLWH [40, 41]. As rK39 is characteristically associated with active disease [42, 43], a sample composed predominantly of asymptomatic individuals is expected to have a lower positivity. Furthermore, even considering the diagnosis of symptomatic disease, the sensitivity of ELISA using the rK39 antigen is considerably worse in PLWH [44]. Therefore, although it can be possible to detect this antigen in asymptomatic individuals [38], these characteristics can limit its use in this context. In the general population, the sensitivity and specificity of recombinant K28 antigen (99.6% and 95-100%, respectively) is similar to rK39 ELISA for the diagnosis of VL [45]. rK28 antigen can be used in rapid tests with good performance, showing high specificity (near 100%) and sensitivity (92%) for the diagnosis of visceral leishmaniasis [46]. However, it has not been used in studies including people from Latin America, and there are few studies discriminating its performance in PLWH. Silva et al [47], using two immunochromatographic tests to detect the antibodies anti-rK39 and anti-rK28, found sensitivities of 67.74 and 61.29%, respectively, in a group with active visceral leishmaniasis and HIV infection, showing poor performance when compared with active visceral leishmaniasis without HIV infection. In our study, in a group of asymptomatic individuals, rK28 ELISA positivity was greater than rK39 ELISA positivity, which can suggest a good perspective of its use in HIV-infected Latin American asymptomatic patients, perhaps as an early marker of Leishmania infection.
In our sample, no individual had a positive DAT. This test is one of the most widely used diagnostic tests for VL around the world [48], and in comparison with other serological methods, it has a good performance in PLWH [30]. DAT can be used to diagnose active forms of the disease and can diagnose infections before the clinical presentation, with results declining to negative values one year after the cure [49]. There are two important points to highlight about the population included in this study. First, the individuals included were predominantly asymptomatic, and nobody had a confirmed leishmaniasis diagnosis or a previous history of this disease. The performance of this test considering this scenario had never been evaluated, probably because there is no gold standard to compare this test with other tests and to establish sensitivity and positivity. Moreover, although a part of the cohort had lived in endemic areas in the past, all subjects were currently living in a non-endemic area for VL. We believe the fact that we are analysing a sample composed of asymptomatic HIV-infected patients not continuously exposed to Leishmania can explain this result, as the positivity of the test depends on the presence of active disease, recent cure or exposure to antigen.
Regarding molecular methods, we used two different targets to perform polymerase chain reaction (PCR). In a systematic review and meta-analysis, de Ruiter et al [50] found a pooled sensitivity and specificity of PCR in peripheral blood of 93.1% and 95.6%, respectively. There are studies demonstrating promising results in immunosuppressed patients as well [51, 52]. Although kDNA is more commonly used for DNA amplification of Leishmania because of the high number of copies per parasite [53], in this study, ITS-1 PCR had greater positivity (4.2%) compared to kDNA PCR (1.7%). Several different genomic targets are used for Leishmania spp. detection, and there is no consensus about the best one, especially due to the discrepancies of objectives and methods of each study [53]. The fact that no patient was positive by PCR and the discrepancy between the two tests reinforces this limitation and the necessity of further studies about this theme, including symptomatic and asymptomatic subjects. Molecular methods have been developed with the purpose of complementing and creating alternatives for the diagnosis of leishmaniasis, as well as the possibility of follow-up and laboratory confirmation of cure [53, 54]. It is possible that more immunosuppressed individuals present a greater periodicity of parasitic circulation in their body. The higher positivity of this method in this type of patients may indicate that this target is more effective in identifying intermittent parasitaemia in this group of patients and that PCR can be used in the future to screen individuals at risk of developing manifest leishmaniasis. This information would be of extreme importance for these individuals with advanced immunosuppression, who are known to present with more severe forms of the disease, more complications and higher mortality [55, 56].
The presence of signs/symptoms did not demonstrate an association with test positivity. First, information about cities with autochthonous transmission was obtained through records of the Ministry of Health of Brazil, and a considerable portion of these cities were considered to have autochthonous transmission due to the presence of just one or a few cases of confirmed VL. Therefore, many participants may not have been exposed to VL, despite living in transmission areas. In the same way, it was not possible to guarantee that, at the moment the individual lived in an endemic area, autochthonous VL was present. Most cases of HIV infection in Brazil are located in the South and Southeast Region, where Sao Paulo state is located. Regarding VL, between 1999 and 2013, 2.328 autochthonous cases of visceral leishmaniasis were confirmed in Sao Paulo state, corresponding to 80 cities with VL transmission. Of these, 202 patients died, corresponding to a lethality of 8.7%. Of note, Sao Paulo city has had no report of autochthonous VL transmission to date [57].
Injecting drug use is the major cause of HIV infections in Leishmania/HIV coinfection in many parts of the world [1]. In Brazil, this pathway of HIV transmission is not as common, and sexual transmission corresponds to more than 95% of HIV infection acquisition [58]. Thus, although we do not have data about the percentage of injecting drug users in the sample, we consider the possibility of Leishmania acquisition through this route to be very low. Concerning the presence of symptoms, 17 of 28 participants presenting with a symptom defined as suggestive of VL had other diseases, including tuberculosis as the most common. Therefore, some of these symptoms were probably more attributable to these diseases than to the diagnosis of VL or to other diseases not yet diagnosed. Therefore, there was no association with this variable and positivity of diagnostic tests.