Evaluation of HIV-Leishmania co-infection in patients from the northwestern Paraná State , Brazil

Leishmaniasis occurs throughout the world and is one of the opportunistic infections that attack HIV-infected individuals. Few data are available on American cutaneous leishmaniasis (ACL) in HIV-infected patients. Current research investigates the occurrence of HIV-Leishmania co-infection in HIV-infected individuals in an endemic region in Southern of Brazil. A non-randomized transversal investigation, molecular and serum epidemiologic type, on the occurrence of ACL in 169 HIV-infected patients was undertaken. The patients were followed up at the Integrated Nucleus of Health of the city Maringá, Southern of Brazil. Results showed that 13 (7.7%) of the HIV-infected patients also presented Leishmania (Viannia) DNA, detectable in blood by PCR. Serology, direct research, culture and PCR in skin material produced negative results. PCR positiveness for Leishmania was not associated with CD4 T lymphocytes count, opportunistic disease, treatment, use of proteases inhibitors, tattooing/piercing or use of injectable drugs, residential environment or previous ACL history. Results show that HIVinfected patients who live in endemic areas may reveal Leishmania DNA in the blood without any ACL symptoms. Above findings may be attributed to anti-retrovirus medicine that controls viral replication and maintains the functionality of the immune system and to a possible antiLeishmania activity of these drugs.


Introduction
American cutaneous leishmaniasis (ACL) is an zoonotic disease caused by protozoa of the genus Leishmania.Infections in humans may either be nonapparent or may display a clinical spectrum ranging from localized, sometimes self-healing cutaneous lesions, to severe mutilating mucocutaneous lesions, or diffuse cutaneous leishmaniasis (AKILOV et al., 2007).ACL is widely distributed in Brazil, from the Amazon region to southern Brazil, with most cases caused by Leishmania (Viannia) braziliensis or Leishmania (Leishmania) amazonensis (ALVAR et al., 2008).
Leishmania protozoa are obligate intracellular parasites, and reside within the mammalian host as amastigotes in phagocyte cells, such as macrophages, dendritic cells and neutrophils.Although the Leishmania species that cause infection in humans induces strong humoral responses, with production of Acta Scientiarum.Health Sciences Maringá, v. 33, n. 1, p. 19-24, 2011 significant levels of antibodies, they appear to play no protective role; in fact, they are rather associated with non-healing forms of leishmaniasis (TRIPATHI et al., 2007).The control of Leishmania infection is mediated by a Th1-type immune response, and experimental studies in murine models of cutaneous leishmaniasis have established a clear dichotomy between Th1mediated protection and Th2-mediated disease susceptibility (SACK; NOBEN-TRAUTH, 2002).The CD4 subset of Th1 cells secrete activators of cellmediated immunity such as IFN-, which induces the production of nitric oxide in phagocytic cells which, in its turn, leads to the parasite's destruction.
Leishmaniasis have been characterized in HIV-1 infected individuals, who may present multiple cutaneous lesions, resembling the picture of diffuse leishmaniasis more frequently found in immunosuppressed patients (RABELLO et al., 2003).HIV-1 infection progressively impairs the immune system function and facilitates the establishment of opportunistic pathogens (CRUZ et al., 2006).
Cases of Leishmania/HIV co-infections have been reported in various parts of the world and most coinfections involve leishmaniasis visceral type (ALVAR et al., 2008).Moreover, recurrence of cutaneous Leishmaniasis has been observed in HIV-1-infected patients with only moderate immunosuppression (COUPPIE et al., 2004).Skin lesions have been reported in HIV-infected patients since the first descriptions of the disease (CARDOSO et al., 2002).Cutaneous forms of the disease with disseminated lesions, involving the nasal, oral or pharyngeal mucosa, have been observed in patients with advanced stage Aids, sometimes accompanied by extensively destructive lesions (FERREIRA;BORGES, 2002).In fact, the above reveal the importance of studies on HIV-Leishmania co-infection.Current research investigates the occurrence of HIV-Leishmania coinfection in HIV/Aids-infected individuals in an ACL endemic region in southern Brazil.Biological samples: A sample of blood was collected from all patients by venous puncture and a fraction of samples was used to obtain the plasma for serologic tests.Skin samples were obtained of the patients with lesion, scars and skin spots (small mark different in colour, texture from the surface): the material of the border of the lesion was collected by scrapping, using a metal scraper free from DNA, and distributed in two tubes free from RNAses and DNAses with 100 μL STE buffer (10 mM TRIS; 1 mM Na 2 EDTA.H 2 O; 0.1 M NaCl; pH 8.0) and were stored at -20 o C, for posterior DNA extraction.

Study of population
Serological Reactions: Indirect immunofluorescence assay (IFA) to ACL was carried out with promastigote forms of L. (V.) braziliensis (SILVEIRA et al., 1999) and for Chagas's disease with Trypanosoma cruzi, strain Y (Imuno-cruzi -Biolab) Plasma was diluted as from 1/20, at the ratio of 2, and a human anti-immunoglobulin G conjugated with fluorescein was employed.Titers ≥ 40 were considered significant for ACL and ≥ 80 for Chagas' disease.
Search of Leishmania sp.: Skin samples were used for: (a) the preparation of smears, stained by Giemsa stain, and then examined for the presence of amastigote; (b) culture in BBA (Blood Base Agar, Difco) medium with antibiotics, incubated at 25ºC and weekly observed during a month for amastigote of Leishmania sp.
Viral load and Counting of T CD4 Lymphocytes: Patients were analyzed and followed up according to the Brazilian Health Ministry protocol (BRASIL, 2007).The HIV viral load followed RT-PCR methodology (COBAS Amplicor hiv-1 MONITOR Test, Roche).T CD4 lymphocytes were counted by flow cytometry with FACSCount Reagents (Becton Dickinson).
Statistic Analysis: The association of opportunistic diseases with T CD4 lymphocyte counts was analyzed by chi-square test with Statistica 6.0.A probability value p < 0.05 was considered statistically significant.

Results
One hundred and sixty-nine HIV-infected patients, 93 males (55%) and 76 females (45%), age ranging between 20 and 67 years old, predominantly between 40 and 57 years (41.4%) were analyzed.PCR showed Leishmania (Viannia) DNA in 13 (7.7%) of blood samples (Figure 1) and negative in all the 10 skin samples.The latter had also negative results by direct examination and culture.All HIV-infected patients had IFA for leishmaniasis negative, although one was IFA positive for Chagas's disease, titer 80.Most HIVinfected patients (94.7%) inhabited the urban area, of whom 165 (97.6%) did not have a previous ACL history.Four of them reported that at least 15 years a ago they had ACL which was treated with pentavalent antimony drugs.However, the thirteen HIV infected patients with DNA Leishmania were among those without any previous ACL history (Table 1).However, PCR positiveness for Leishmania was not associated with CD4 T lymphocytes count (p = 0.7799), opportunistic disease (p = 0.3812), treatment (p = 0.9225), residential environment (p = 0.8047) or previous ACL history (p = 0.7150), use of proteases inhibitors (p = 0.2315), tattoo/piercing (p = 0.3232) or use of injectable drugs (p = 0.3290).It has also been reported that 98 (58.0%) of HIV/Aids infected patients had one or two opportunistic diseases.Cytomegalovirosis was the most frequent with 128 cases, followed by toxoplasmosis with 61 cases, by candidiasis with 28 cases, by herpes zoster with 20 cases, and by tuberculosis with 11 cases.PCR was positive for six toxoplasmosis-infected patients, one with toxoplasmosis and herpes zoster, two with herpes zoster, one with tuberculosis and three with no opportunistic disease (Table 2).

Discussion
Results show that 7.7% of HIV/ds-infected patients also had Leishmania (Viannia) DNA in their blood, detected by PCR.All the patients had negative results in serology and in direct search, culture and PCR of the skin samples.ACL is endemic in the northwestern region of the Paraná State, Brazil.From the total cases of the southern region of Brazil, 97% occurred in Paraná State (BRASIL, 2007).According to Silveira et al. (1999), 69.3% of the 804 ACL-positive patients were rural workers or inhabited the rural area; in this area L. (V.) braziliensis is the main agent of ACL.Current investigation, however, reported that only 5.3% of HIV/-infected patients lived in the rural areas.Moreover, 13 patients with DNA Leishmania in their blood were from the urban region.Only 10 infected patients having lesion, scars and skin spots, although direct research and PCR produced negative results in all of them.
Serological tests may be particularly useful in the diagnosis of visceral or mucocutaneous leishmaniasis (DENIAU et al., 2003).In this work IFA was employed to investigate anti-Leishmania serological reactivity even though in a previous investigation within the same region the technique had 75.7% sensitiveness (SILVEIRA et al., 1999).Since results show that infected patients did not have anti-Leishmania antibodies at detectable levels by IFA, it is necessary that more sensitive techniques must be employed.In current research, a single patient had positive serology for Chagas's disease.
Specific primers used in the PCR for Leishmania (Viannia) amplify the preserved region (69 bp) of the k-DNA mini-circle and detects 0.9 fg of the parasites' DNA (VELÁSQUEZ et al., 2006).Since dermatological manifestations in HIV-infected patients have atypical clinical traits, in current research every type of skin lesion, even tattoos and spots, were suspected of ACL.In fact, reports are extant with regard to amastigotes in health skin, tattoos, Kaposi's sarcoma lesions and herpes zoster (PUIG;PRADINAUD, 2003).However, only one out of 30 HIV-infected patients with tattoos, skin spots or piercing had PCR positive.Further, the use of injectable drugs was also taken into account since reports give a high predominance of Leishmania infantum in syringe-sharing intravenous drug users (CRUZ et al., 2002).Although 12.4% of patients declared themselves drug users, no one was ACL positive.
A significant decrease in HIV-Leishmania coinfection has been reported in France (DEL GIUDICE et al., 2002) and in Spain (DE LA ROSA Acta Scientiarum. Health Sciences Maringá, v. 33, n. 1, p. 19-24, 2011et al., 2002) after the introduction of anti-retroviral therapy, which may actually be interpreted as a reconstitution of the immune system.Besides, "in vitro" studies in L. major and L. infantum cultures showed that the proteases-inhibiting drugs (Indinavir and Saquinavir) have leishmanicidal activity (SAVOIA et al., 2005).Current research has shown that in the context of HIV-infected patients under analysis, 70.4% were undergoing treatment, of which 44.5% used protease inhibitors, including the four subjects who had had ACL.Although studies on the drugs' anti-Leishmania (Viannia) activity have not been undertaken, such possibility may not be disposed of and may be investigated later on.It has been found that 58.0% of HIV/Aidinfected subjects had one or more opportunistic diseases, among whom 10 subjects that presented Leishmania DNA.Opportunistic infections may accelerate HIV infection by the microorganisms' capacity in stimulating the Th2 cytokines (IL-4, IL-10, among others) production which may trigger the progression of HIV/AIDS disease and it has already been demonstrated that HIV/Leishmania co-infection favors viral replication and worsens the immunosuppression state (FERREIRA;BORGES, 2002).The immunosuppression may cause an increase in viral load, decrease in the number of T CD4 + cells and an uncontrolled multiplication of Leishmania.However, in current studies no significant association was found between any investigated opportunistic infections and PCR positiveness.

Conclusion
Results show that HIV-infected patients that inhabit endemic areas may have Leishmania DNA in their blood without triggering the humoral immune response and without any ACL symptoms.A possible explanation could be that the blood DNA detected by PCR methods in asymptomatic patients would be a fossil DNA, without the ability to trigger the immune system.On the other hand, the anti-retroviral treatment, which is able to control viral replication, can maintain the functionality of the immune system and control of Leishmania infection, or the drug may have activity against Leishmania.However, the reasons for these findings are not clear, but the evidence of Leishmania DNA in blood found in subjects with no history of ACL raises questions concerning their clinical consequences.Since that in Brazil antiretroviral treatment is indicate for all HIV-infected patients, symptomatic or not, who have a T CD4 + lymphocyte count lower than 350 cells μL -1 , it is very important that all HIV-infected patients living in the Leishmania-transmission area are analyzed for ACL.