There are few studies evaluating the prevalence of Leishmania asymptomatic infection among HIV-infected individuals, and there are no studies analyzing 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 results were smaller than those reported by Orsini et al., 20.2% [10]. In another study performed in Brazil, Carranza-Tamayo et al. showed similar data, with an overall prevalence of 16.0%, [9]. Considering studies in other parts of the world, the frequency is lower than that found by Garrote et al., 64.0%, in Spain [23]. These discrepancies are probably explained by the use of different diagnostic tests, including different antigens and molecular targets. Recently, in a systematic review about asymptomatic visceral leishmaniasis in the Indian subcontinent, Hirve et al. [24], including 31 articles and using 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. [16], in Marroco, found prevalence of 5% in HIV-infected patient, 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 full-blown symptomatic disease, there is little data in Latin America regarding the performance of diagnostic methods [25].
Data also demonstrated poor agreement between serological tests. This fact has been shown by other prevalence studies involving HIV infected patients and immunocompetent individuals [9, 10, 26, 27], which can be accounted for the use of different antigens and molecular targets (surface, soluble or recombinant antigens) that can detect many stages of the asymptomatic infection.
ELISA using Leptomonas seymori antigen was performed for the first time in patients with HIV infection. It has already been demonstrated that this antigen had a good performance for the diagnosis of visceral leishmaniasis; in fact, its performance is comparable to L. chagasi antigen. Similarly to other crude antigens, there is the possibility of cross-reactivity with T. cruzi and other Leishmania species [18]. Recently, Kesper et al. [28], 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 patients did not have HIV-infection. We support strongly that further studies are necessary to determinate the sensitivity and specificity for VL diagnosis in individuals with HIV infection, using Leptomonas antigen.
IFAT using L. major-like antigen is a test recommended by the Ministry of Health of Brazil for the diagnosis of leishmaniasis. In this study, IFAT showed greater positivity compared to ELISA Leptomonas. In other studies evaluating the prevalence of Leishmania/HIV coinfection, ELISA using crude antigens demonstrated greater positivity compared to IFAT [9, 10], and this was attributed to the fact that IFAT detects mainly antibodies against surface antigens, while crude antigens ELISA detect a wider variety of antibodies directed against soluble antigen components. In our study, this difference was not significant and probably related to divergence between specificity of these tests than to the immunological characteristics of antigens.
Considering only serological methods, we found that ELISA Leptomonas and IFAT had positivity associated with T CD4+ counts less than 200 cells/mm³. This data can be, at first, considered surprising, given that serological methods have worse sensitivity for the visceral leishmaniasis diagnosis in HIV infected patients compared to the immunocompetent individuals [6, 25, 29]. 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 [3]. It is important to highlight that all this information is related to the performance of diagnosis tests in subjects with full-blown symptomatic visceral leishmaniasis and, herein, we are discussing 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 T CD4 + counts had subclinical or latent diseases, which may have interfered with the results. Another question is the possible variability of humoral response in HIV infected subjects. Gradoni et al [30], comparing serological IFAT titles of HIV infected and non-HIV infected individuals with visceral leishmaniasis, showed worse sensitivity of this method in those infected with HIV, but a greater variability of titles in this group, with some patients 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 that acquiring Leishmania infection before HIV infection should produce high levels of IgG directed against antigens recognized before T cell impairment due to the HIV. This response is probably related to the increased IgG production because of non-specific polyclonal B cell activation, which occurs more frequently in patients with severe immunosuppression [3, 30]. This abnormal humoral response, more evident in severe 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.
ELISA rK39 had the lowest positivity comparing all serological methods. This antigen has been used both in ELISA assays and in rapid diagnostic tests [31–33], including in HIV infected patients [34, 35]. As it is characteristically associated with active disease [36, 37], a sample composed predominantly by asymptomatic individuals is expected to have a lower positivity. Furthermore, even considering the diagnosis of full-blown symptomatic disease, the sensitivity of ELISA using rK39 antigen is considerably worse in HIV-infected individuals [38]. Therefore, although it can be possible to detect this antigen in asymptomatic individuals [32], these characteristics can limit its use in this context. In the general population, the sensitivity and specificity of recombinant antigen k28 (99.6% and 95–100%, respectively) is similar to ELISA rK39 for the diagnosis of VL [39]. This antigen can be used in rapid tests with a good performance, showing high specificity (near 100%) and sensitivity (92%) to the diagnosis of visceral leishmaniasis [40]. However, it has not been used in studies including patients in Latin America, and there is few studies discriminating its performance in HIV-infected subjects. Silva et al [41] using two immunochromatographic tests to detection antibodies anti-rK 39 and anti rK28 found sensitivity of 67.74 and 61.29% respectively, in patients 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, its positivity was greater than ELISA rk39, which can suggest a good perspective of its use in HIV infected Latin American asymptomatic patients, maybe 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 the diagnosis of VL around the world [42] and, in comparison with other serological methods, it has a good performance in HIV infected patients [25]. DAT can be used to diagnose active forms of the disease and can diagnose infections before the clinical presentation, with titles declining to negative values one year after the cure [43]. There are two important points to highlight in the population included in this study. Firstly, 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 one with other tests and to establish sensitivity and positivity. Moreover, although a part of 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 analyzing a sample composed by asymptomatic HIV-infected patients not continuously exposed to Leishmania can explain this result as the positivity of test depends on the presence of active disease, recent cure or exposition to antigen.
Regarding molecular methods, we used two different targets to perform the polymerase chain reaction (PCR). In a systematic review and meta-analysis, de Ruiter et al [44] 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 [45, 46]. Although kDNA is more used for DNA amplification of Leishmania, because of the high number of copies per parasite [47], 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 sp. detection, and there is no consensus about the best one, especially due to the discrepancies of objectives and methods of each study [47]. The fact that no patient had both PCR positive and the discrepancy between 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 control of cure [47, 48]. Possibly, more immunosuppressed patients present a greater periodicity of parasitic circulation in their organism. The higher positivity of this method in this context of patients may indicate that this target is more effective in identifying intermittent parasitemia in this group of patients, since it can be used in the future to screen individuals at risk of developing manifest leishmaniasis. This information would be of extreme importance for these patients with advanced immunosuppression, who are known to present with more severe forms of the disease, more complications and higher mortality [49, 50].
Social factors or the host may constitute a risk for the occurrence of Leishmania infection. Here, we evaluated aspects related to the host, as risk factors to Leishmania infection. Presence of signal/symptoms did not demonstrate association with test positivity. At first, the information about cities with autochthonous transmission was obtained through records of the Ministry of Health of Brazil showing that a considerable part of these cities is considered having autochthonous transmission due to the presence of just one or few cases of confirmed VL. Therefore, many patients may not have been exposed to VL, despite living in transmission areas. In the same way, it was not be possible to guarantee that, at the moment the individual lived in an endemic area, this place presented autochthonous VL. 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 evolved to death, corresponding to a lethality of 8.7%. Of note, Sao Paulo city has had no report of autochthonous VL transmission to date [51].
Injecting drug use is the major drive of HIV infections in many parts of the world, in Leishmania/HIV coinfection [1]. In Brazil, this pathway of HIV transmission is not important and sexual transmission corresponds to more than 95% of HIV infection acquisition [52]. Thus, although we do not have data about the percentage of injecting drugs 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 patients presenting with some symptom defined as suggestive of VL had other diseases, including tuberculosis as the most cited. Therefore, probably part of these symptoms was more attributable to these diseases than to diagnosis of VL or to other diseases not diagnosed yet. Due to that, there was not association with this variety and positivity of diagnostic tests.