Comparison of laboratory methods for diagnosis of human visceral leishmaniasis in the Center-West Region of São Paulo State, Brazil

. Visceral leishmaniasis (VL) is a neglected disease and represents an important public health problem. The present study aimed to compare the conventional laboratory methods with the molecular method for VL diagnosis in human blood samples. Direct parasitological test of bone marrow aspirate (direct parasitological test), rapid immunochromatographic test (RIT), indirect immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR) were used to evaluate 26 human samples with clinical suspicion of VL. The positivity rates obtained for direct parasitological test, RIT, IFAT and PCR were 80.8%, 80.8%, 73.1% and 84.6%, respectively. PCR had greater positivity and agreement with the other methodologies. Therefore, the use of PCR in the diagnostic routine should be encouraged to complement the serological results.


Introduction
Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania and represents a group of distinct diseases, including visceral leishmaniasis (VL) and American tegumentary leishmaniasis (ATL) (Alvar et al., 2012).In 2018, more than 95% new cases of VL reported to the World Health Organization occurred in 10 countries: Brazil, China, Ethiopia, India, Iraq, Kenya, Nepal, Somalia, South Sudan and Sudan (WHO, 2020).In Brazil, the major species involved in VL is Leishmania infantum (syn.chagasi) (Brasil, 2006).
Visceral leishmaniasis is a neglected disease and represents an important public health problem.The Brazilian program for VL control recommends clinical and laboratory diagnosis, treatment of infected individuals, identification and control of domestic reservoirs, control of vectors, environmental management and health education services (Brasil, 2006).Nevertheless, VL is endemic, leading to autochthonous cases in 25% municipalities in 21 of 26 Brazilian states.According to the last update in 2021, Brazil notified 50,270 cases from 2006 to 2020, accounting for a mean of 3,351 cases per year (WHO, 2021).
Diagnosis of leishmaniasis is obtained by an association among clinical, epidemiological and laboratory information.Conventional laboratory methods have been used, including parasitological and serological tests; however, they have limitations, while molecular methods have increasingly been explored and adopted as a complement and/or an accurate diagnostic alternative (de Paiva-Cavalcanti et al., 2015;Ghasemian et al., 2016).Polymerase chain reaction (PCR) is a highly sensitive and specific methodology which can be adopted for both diagnosis and treatment follow-up, constituting a useful diagnostic tool for low parasitic load, HIV co-infection and relapsed and asymptomatic cases (Reithinger & Dujardin, 2007;Fraga et al., 2010;Lindoso, Cunha, Queiroz & Moreira, 2016;Hossain et al., 2017).
The region of Bauru, located in the Center-West part of São Paulo State, Brazil, is endemic for VL and is considered a regional pole that plays an important role in the dissemination and expansion of this disease (Cardim et al., 2013); such high incidence indicates the need of studies comparing the different diagnostic methods and evaluating the possible inclusion of other methodologies.Thus, the current study aimed to compare the conventional methods used for VL diagnosis in human blood samples with the molecular method.

Ethical statement
The present study was approved by the Research Ethics Committee of Adolfo Lutz Institute No. 1.131.447.

Samples
Visceral leishmaniasis is a mandatory notifiable disease.In Brazil, every clinical suspicion of VL requires the control program to immediately notify this patient through a specific form of the Brazilian Notifiable Diseases Information System.The local Surveillance Department collects all clinical and epidemiological data to investigate the case.During this process, additional information regarding diagnosis, treatment and results are included.For diagnosis, the human sample is forwarded to and analyzed at the IAL, based on a previous suggestive clinical condition of VL.Twenty-six human cases with clinical suspicion of VL were selected to perform routine laboratory methods, direct parasitological test of bone marrow aspirate, rapid immunochromatographic test (RIT) and indirect immunofluorescent antibody test (IFAT), as well as complementary methodology, polymerase chain reaction (PCR).The study did not include patients with relapsed and/or HIV co-infection, but only cases with suspected primary infection.

Direct parasitological test
Direct parasitological test was performed for bone marrow aspiration puncture swabs colored with Giemsa.Slides were visualized with 100x immersion oil objective under an optical microscope to confirm the presence of amastigote forms in the tissue.
Rapid immunochromatographic test (RIT) The RIT adopted for human serum samples was IT LEISH ® (Biorad Laboratories, Marnes-la-Coquette, France), which uses recombinant K39 antigen to detect antibodies to Leishmania spp.(Assis et al., 2008).The tests were performed according to the manufacturer's recommendations.

Indirect immunofluorescent antibody test (IFAT)
To perform IFAT, the human Leishmaniasis IFAT Kit (Bio-Manguinhos, Rio de Janeiro, RJ, Brazil) was used to detect antibodies to Leishmania spp. in human serum with clinical suspicion of VL, according to the manufacturer's recommendations (Brasil, 2006).Slides were visualized under a fluorescence microscope (Olympus BX51, JPY).According to the Brazilian program for VL control, positive titration equal or superior to 1:80 and 1:40 titers is considered indeterminate, and the test should be repeated after 30 days (Brasil, 2006).

Molecular analyzes DNA extraction and quantification
DNA extractions from blood samples were performed with the Illustra blood genomic Prep Mini SpinKit (GE Healthcare, Pittsburgh, USA), as instructed by the manufacturer.Quantification was evaluated in a spectrophotometer (Epoch-Biotek, USA).

Electrophoresis on agarose gel
An 8-μl aliquot of each amplified product was added of 2 μl run buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol, 70% Milli-Q water) and homogenized.The adopted molecular marker was 4 μl 100 bp ladder (Invitrogen, USA).The amplified material was visualized by electrophoretic run on 1.5% agarose gel added of 0.1 μl/mL SYBR Safe DNA gel stain (Invitrogen, USA).The electrophoretic run was performed in a horizontal vessel containing 1X TBE (89 nM Tris-HCl, 89 mM boric acid and 20 mM EDTA), at 65V, for 60 minutes.After the end of the run, the amplified bands were visualized under ultraviolet light (296 nm) and recorded with a transilluminator (Syngene, USA); the image was captured by the digital documentation system.

Results and discussion
Of the 26 samples analyzed, 21 (80.8%) were positive by direct parasitological test of bone marrow aspirate, 21 (80.8%) were positive by RIT,19 (73.1%) were positive by IFAT with titration equal to 80, six (23.1%) were indeterminate by IFAT with titration equal to 40, and 22 (84.6%) were positive by PCR.The level of agreement between methodologies performed for VL diagnosis was good and excellent for direct parasitological test, RIT and PCR.However, IFAT showed less positivity and moderate to weak agreement with the other methodologies, possibly due to its indeterminate results (Table 1).Parasitological examination is considered a "gold standard" for the diagnosis of leishmaniasis, although lower and more variable sensitivity has been described depending on the used sample (Srivastava, Dayama, Mehrotra & Sundar, 2011;Muñoz et al., 2016).In the current study, this method allowed the identification of 80.8% individuals with VL.Several studies have demonstrated that the sensitivity of molecular methods is higher than that of parasitological examination.In addition, the molecular method has been used for patients with negative results by the parasitological test (Osman et al., 1997;Romero et al., 2001;Antinori et al., 2007;Fraga et al., 2010).Piarroux et al. (1994) analyzed 73 peripheral blood samples from patients with suspected VL and found that PCR had a high sensitivity (82%), compared to parasitological test (55%) and culture (55%).
Among the serological tests performed in humans, RIT (rK39) allowed identification of 80.8% individuals with VL.Recombinant K39 antigen is described as a very useful tool for VL diagnosis, but its performance may vary according to the applied region due to the parasite diversity and the individuals' different antibody concentrations, age, nutritional status and immunological response (Cunningham et al., 2012).In Brazil, its sensitivity has been described between 84.7 and 93% (Guimarães, Lemos, Corey, & Dietze, 2003;Assis et al., 2008;Cunningham et al., 2012).However, according to da Silva et al. ( 2018), there is a decrease in sensitivity from 95.45% to 61.29% for patients co-infected with HIV.Ozerdem, Eroglu, Genc, Demirkazik & Koltas (2009) compared PCR, RIT (rK39) and parasitological tests in 50 peripheral blood samples from patients with suspected VL in Turkey and demonstrated that PCR is the most sensitive technique.
IFAT obtained the lowest positivity and agreement with the other methodologies, possibly due to the presence of indeterminate results.Its performance is associated with the disease time, clinical form, involved species, and used antigenic preparation (Brasil, 2006;Assis et al., 2008;Barroso-Freitas et al., 2009).The major disadvantage of IFAT is the possibility of cross-reactions with other diseases, especially Chagas disease (Brasil, 2006;Pedras, de Gouvê Viana, de Oliveira & Rabello, 2008).Several authors have suggested that it should be replaced and/or complemented with other serological tests or molecular methods, which are considered more sensitive and specific (Neitzke-Abreu et al., 2013;Machado de Assis, Azeredo-da-Silva, Werneck & Rabello, 2016).
In the current study, the high positivity of PCR to detect the genus Leishmania was demonstrated in 84.6% samples from patients with clinical signs of VL.Another study reported 91.3% positivity for PCR and realtime PCR in 100 peripheral blood samples from patients with clinical signs of VL (da Costa Lima et al., 2013).In addition, Fraga et al. (2010) proved PCR is suitable for diagnosing VL in children since it is sensitive and safe and involves less invasive tissue collection, compared to other tests.
Several authors have demonstrated that diagnosis of VL is decisive in reducing morbidity and mortality rates (da Costa Lima et al., 2013;Martins-Melo, Lima, Ramos Jr, Alencar & Heukelbach, 2014).Therefore, all efforts must be made to improve the diagnosis of such an important public health disease, including the adoption of more accurate and affordable methods, since PCR, due to its costs, is not generally available in resource-limited settings.

Conclusion
In the present study, PCR obtained greater positivity and agreement with the other analyzed methodologies.Therefore, the use of PCR in routine diagnosis of human VL should be encouraged to complement serological results.
Adolfo Lutz Institute (IAL) is a National Laboratory on Public Health, which focuses on diagnostic, sanitary and epidemiological surveillance.The Institute, in Bauru region, operates in 38 cities in the Center-west part of São Paulo State and attends several Programs such as Visceral Leishmaniasis Surveillance and Control Program (VLSCP).The analyzed human samples derived from cities covered by the Institute.

Table 1 .
Kappa coefficient of agreement with 95% confidence interval, according to the laboratory methods performed for the diagnosis of human visceral leishmaniasis.RIT = rapid immunochromatographic test; b direct parasitological test = direct parasitological test in bone marrow aspiration puncture; c IFAT = indirect immunofluorescent antibody test; d PCR = polymerase chain reaction. a