International Journal of Infectious Diseases 95 (2020) 44–49

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Predictors of relapse of visceral leishmaniasis in inner

São Paulo State, Brazil
José Cláudio Simãoa , Cassiano Victóriab , Carlos Magno Castelo Branco Fortalezaa,c,*
a
Postgraduate Program in Tropical Diseases, Botucatu Medical School, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil
b
Department of Veterinary Hygiene and Public Health, Veterinary Faculty, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil
c
Department of Tropical Diseases, Botucatu Medical School, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: Objectives: Visceral leishmaniasis (VL) is a public health threat for several tropical countries, including
Received 29 December 2019 Brazil. Therapy failures and relapses aggravate VL morbidity and mortality. Our study aimed at
Received in revised form 12 February 2020 identifying predictors of relapse and thus contributes to directing therapeutic options and patient follow-
Accepted 14 February 2020
up.
Methods: A nonconcurrent cohort of 571 subjects who completed successful therapy for VL in the city of
Keywords: Bauru, São Paulo State, Brazil, was followed for 24 months in order to identify the incidence and
Visceral leishmaniasis
predictors of relapse. Extensive review of medical charts and laboratory files was conducted. Univariate
Predictors of relapse
Visceral leishmaniasis-HIV coinfection
and multivariable Cox regression models were used to identify predictors for the outcome of interest. A
hierarchical strategy was used for variable selection in multivariable models.
Results: Relapses occurred in 6.8% of treated subjects, after a median of 6 months (interquartile range, 4–
9). In a comprehensive multivariable model, relapse was associated with: HIV-coinfection (hazard ratio
[HR], 7.47; 95% confidence interval [CI], 2.58–21.55); the presence of lower limb edema (HR, 6.06; 95%CI,
1.38–26.77) and low platelet count upon admission (HR for platelet count  1000, 0.99; 95%CI, 0.98–0.99)
; and secondary pneumonia (HR, 5.49; 95%CI, 1.49–20.18). On the other hand, therapy with Liposomal
Amphotericin (as opposed to Antimoniate) was not independently associated with relapse (HR, 5.97; 95%
CI, 0.63–56.29).
Conclusion: Besides reinforcing the impact of HIV coinfection on the outcome of VL, our study points to
clinical and laboratory findings that characterize patients who were more likely to relapse. Those groups
should be more closely followed, and possibly could benefit from novel therapeutic options.
© 2020 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

Introduction
Visceral leishmaniasis (VL) is a public health threat for several
tropical countries, including Brazil (Antinori et al., 2012; Burza
et al., 2018). In the past two decades, Brazil witnessed changes in
the epidemiology of VL, which moved from a rural endemic in
poorest Northeastern States to an urban emerging disease
spreading into the more developed Southeastern Region (Conti
et al., 2016; Martins-Melo et al., 2018). In this new setting, the
epidemics of VL and AIDS intertwined (Lindoso et al., 2014; Leite de
Sousa-Gomes et al., 2017; Lindoso et al., 2018). Governmental
guidelines for the therapy of VL and VL-HIV coinfection were
expedited, but data concerning their efficacy are still limited (de
Melo and Fortaleza, 2013).
It is worth noting that factors associated with death among VL
patients (including therapeutic failure) have been far more
investigated than those affecting the likelihood of relapse (Belo
et al., 2014). With that in mind, we conducted a study aimed at
quantifying relapse and identifying clinical and laboratory
predictors for that outcome.
Methodology
Study design and setting

We studied a nonconcurrent cohort of patients with laboratory

* Corresponding author at: Departamento de Doenças Tropicais, Faculdade de
Medicina de Botucatu, UNESP, Distrito de Rubião Junior, S/N, 18618-970, Botucatu,
São Paulo, Brazil.
E-mail address: cmfortaleza@uol.com.br (C.M.C.B. Fortaleza).
confirmed VL (either by observation of amastigotes in bone
marrow aspirates or K39-based serological tests) cared for in the
Bauru State Hospital (335 beds). That hospital admitted 97% of VL

https://doi.org/10.1016/j.ijid.2020.02.028
1201-9712/© 2020 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 J.C. Simão et al. / International Journal of Infectious Diseases 95 (2020) 44–49
cases from the city of Bauru (2218'53"S, 49 03'38"W) and several
surrounding municipalities from inner São Paulo State, Brazil. The
first VL cases in that area date from 2003.
Subjects, inclusion and exclusion criteria
Our cohort included all subjects admitted with a confirmed VL
diagnosis from 2003 through 2016 whose therapy was considered
successful, on the basis of relevant improvement in clinical and
laboratory findings, alongside absence of signs and symptoms for
30 days after finishing treatment. Patients who died during the
initial admission, as well as those who were previously treated for
VL, were excluded. Our outcome of interest (relapse) was defined
as a recurrence of signs and symptoms from one to 24 months after
completing successful therapy.
Operational procedures
Extensive review of medical charts and laboratory files was
conducted. We recorded demographic data, comorbidities and
clinical and laboratory findings, as well as the anti-leishmania
therapy instituted (Amphotericin B deoxycholate, Liposomal
Amphotericin B or Antimoniates). It is worth noting that, in
2005, São Paulo state guidelines recommended therapy with
Liposomal Amphotericin for children under 10 years of age, adults
over 50, pregnant, HIV-coinfected and other immunosuppressed
patients (de Melo and Fortaleza, 2013). That guideline was applied
in the study hospital.
Statistical analysis
Statistical analysis of predictors of relapse was performed in
SPSS 20 (IBM, Armonk, NY), using univariate and multivariable
Figure 1. Rationale for including the variables in multivariable Cox regression models for predictors of relapse in patients treated for visceral leishmaniasis in inner Brazil.
45
models of Cox regression. Our choice for a time-to-event model
was intended to avoid biases due to eventual losses of follow-up. In
order to select variables for multivariable models, we used a
hierarchical strategy, entering variables in successive blocks: (1)
demographics; (2) comorbidities; (3) symptoms/signs upon
admission; (4) laboratory finding upon admission; (5) secondary
infections; (6) changes in blood cell counts during therapy; (7)
therapeutic option (Antimoniate or Liposomal Amphotericin B). A
P-value of <0.1 was required for moving one variable into the
following block. However, age group was forced into all models,
regardless of its statistical significance. Furthermore, we studied
models including alternative clinical or laboratory findings. The
rationale for variable selection for multivariable models is
represented graphically in Figure 1. For dealing with missing data,
we anticipated three strategies: (i) excluding cases from analysis if
they represented less than 5% of our sample; (ii) creating a
“missing” category if cases with a relevant missing variable
represented 5% or more of the total sample; (iii) exclude the
variable from the analysis if it was not considered relevant by the
authors.
Results
Among 627 subjects with confirmed VL, there were 56
deaths (mortality rate, 8.9%). The remaining 571 were consid-
ered successfully treated and therefore included in our study
cohort. There were no losses to follow up during the 24-month
period.
There were 38 relapses (overall rate, 6.8%): 15 among HIV-
coinfected patients (rate, 23.7%) and 23 in HIV-seronegative
subjects (rate, 4.5%). That difference was significant at a P < 0.001
level. Overall, relapse occurred after a median interval of 6 months
from therapy (quartiles, 4 and 9 months). It is noteworthy that we
46 J.C. Simão et al. / International Journal of Infectious Diseases 95 (2020) 44–49

included overall 55 HIV-coinfected patients, but other immuno-
suppressed (e.g., transplant recipients) patients were not cared for
in the study period. Also, no pregnant woman was admitted during
the study period.
Results from univariate analysis are available in a supplemen-
tary file (Table 1). Final multivariable models are presented in
Table 2, and survival graphics for relevant categorical variables are
shown in Figure 2. Briefly, Model #1 (not including laboratory
results) found HIV-coinfection, edema of lower limbs and
secondary pneumonia as independent predictors of relapse. In
Model #2 (including laboratory results but not clinical data)
relapse was associated with lower platelet count at admission and
lower platelet count change during therapy. A comprehensive
model (Model #3, including both clinical and laboratory data)
found that HIV-coinfection, edema of the lower limbs, secondary
pneumonia and lower platelet count at admission significantly
predicted relapse.
Importantly, although therapy with Liposomal Amphotericin B
(as opposed to antimoniate) was associated with relapse in
univariate tests, that effect was not detected after adjusting for
confounders (especially HIV co-infection). Also, no difference in
the likelihood of relapse was detected for age groups.

Table 1
Cohort characteristics and univariate results of Cox regression models for predictors of relapse of Visceral Leishmaniasis in the city of Bauru, inner Brazil.

Predictors Cohort characteristics* HR (95% CI) P

Demographic data
Male gender 362 (63.4) 1.45 (0.72–2.92) .29
Age in years 18 (3–43) 1.00 (0.99–1.02) .80
Age groups
0–4 years 174 (30.4) 0.95 (0.47–1.94) .88
5–9 years 60 (10.5) 0.63 (0.18–2.13) .63
10–19 year 64 (11.2) 0.38 (0.09–11.64) .20
20–59 years (reference category) 229 (40.1) ... ...
60 years or older 44 (7.7) 0.45 (0.13–2.44) .45
Schooling for at least 9 years 128 (22.4) 1.23 (0.50–2.54) .57
African–Brazilians 268 (46.9) 0.91 (0.48–1.72) .77
Comorbidities
Heart disease 12 (2.1) 1.25 (0.17–9.13) .82
Lung disease 15 (2.6) 2.06 (0.50–8.57) .32
Renal disease 10 (1.8) 0.05 (0.00->1.000) .58
Liver disease 6 (1.1) 0.05 (0.00–>1.000) .67
Diabetes mellitus 8 (1.4) 0.05 (0.00–>1.000) .62
CNS disease 7 (1.2) 0.00 (0.00–>1.000) .64
Lymphoma/leukemia 0 (0.0) 0.05 (0.00–>1.000) .76
Solid malignancy 4 (0.7) 0.05 (0.05–>1.000) .73
HIV coinfection 55 (9.6) 6.98 (3.64–13.39) <.001
Charlson comorbidity index 1.38 (1.23–1.54) <.001
Clinical picture upon admission
Time of symptoms (weeks) 0.99 (0.98–1.00) .27
Severe paleness** 303 (53.1) 1.57 (038–6.52) .54
Jaundice 33 (5.8) 0.05 ( . . . –17.38) .31
Liver enlargement 417 (73.0) 1.20 (0.57–2.52) .64
Spleen enlargement 430 (75.3) 1.24 (0.53–2.70) .59
Edema of the lower limbs 5 (0.9) 8.57 (2.06–35.63) <.001
Hemorrhagic manifestations** 9 (1.6) 1.75 (0.24–12.77) .58
Laboratory findings upon admission
Hemoglobin (g/dL) 8.2 (7.1–9.4) 0.82 (0.69–0.98) .02
Hematocrit (%) 25.2 (21.8–28.9) 0.93 (0.77–0.98) .005
White cell counts ( 100) 25.1 (17.0–39.0) 0.99 (0.98–1.01) .33
Neutrophils count ( 100) 10.5 (6.5–16.7) 0.98 (0.95–1.02) .35
Platelet count ( 1000) 82.7 (52.4–121.0) 0.99 (0.98–0.99) .04
Serum creatinine (g/dL) 0.9 (0.7–1.1) 0.98 (0.83–1.15) .80
Serum AST (g/dL) 103.0 (55.0–220.0) 1.00 (0.99–1.00) .48
Serum ALT (g/dL) 56.0 (26.0–100.0) 1.00 (0.99–1.00) .48
Alkaline Phosphatase (g/dL) 269.0 (208.0–423.0) 0.99 (0.99–1.00) .16
Gamma–GT (g/dL) 175.0 (72.0–175.0) 0.99 (0.99–1.00) .17
Serum Albumin (g/dL) 2.5 (1.6–3.6) 0.62 (0.27–1.38) .24
Secondary infections upon admission
Overall infections 6 (1.1) 9.56 (2.94–31.09) <.001
Pneumonia 5 (0.9) 12.02 (3.69–49.25) .002
Urinary tract infections 1 (0.2) 0.05 (0.00–>1000) .86
Improvement in blood cell counts***
Hemoglobin change (g/dL) 0.7 (–0.3–2.3) 1.01 (0.96–1.06) .71
Hematocrit change (%) 2.2 (–1.2–7.2) 1.00 (0.99–1.00) .90
White cell counts change ( 100) 12.00 (2.1–24.00) 1.00 (0.99–1.00) .53
Neutrophils count change ( 100) 3.5 (–0.5–10.40) 1.00 (0.99–1.01) .98
Platelet count change (x 1000) 45.0(5.7–113.00) 0.99 (0.99–0.99) .02
Therapy
Amphotericin B deoxicholate 22 (3.9) 0.04 (0.00– >1,000) .79
Liposomal Amphotericin 433 (75.8) 9.84 (1.35–71.71) .02
Therapy with Antimoniates 116 (20.3) Reference category ...

Note. Associations with P < 0.5 are presented in boldface. HR, Hazard Ratio; CI, Confidence Interval.
*
Proportional data presented in number(%), while continuous and discrete data are presented in median (quartiles).
**
Detected by the assistant doctor upon admission.
***
Improvement = values at discharge – values upon admission.
 J.C. Simão et al. / International Journal of Infectious Diseases 95 (2020) 44–49 47

Table 2
Final multivariable models of Cox regression analysis for predictors of relapse in visceral leishmaniasis in Bauru, Southeastern Brazil.

Predictors Model #1 Model #2 Model #3

Age group HR (95%CI) P HR (95%CI) P HR (95%CI) P

0–4 years 2.21 (0.74–6.57) 0.15 1.50 (0.51–4.60) 0.46 1.78 (0.58–5.46) 0.32
5–9 years 1.51 (0.35–6.65) 0.58 1.44 (0.33–6.33) 0.64 1.72 (0.39–7.68) 0.48
10–19 years 2.22 (0.44–11.07) 0.33 2.25 (0.45–11.37) 0.33 2.67 (0.51–14.0) 0.25
20–59 years (reference) ... 0.0.0. ... ... ... ...
60 years and older 0.97 (0.20–3.67) 0.97 0.91 (0.19–4.39) .91 1.10 (0.23–5.43) 0.90
HIV coinfection 7.53 (2.68–21.12) <0.001 8.0 (2.83–22.68) <0.001 7.47 (2.58–21.55) <0.001
Edema of lower limbs 7.714 (1.67–30.60) 0.008 ... ... 6.06 (1.38–26.77) 0.02
Secondary pneumonia 5.87 (1.70–20.24) 0.005 ... ... 5.49 (1.49–20.18) 0.01
Platelet count upon admission (x1000) ... ... 0.99 (0.98–0.99) 0.008 0.99 (0.98–.099) 0.01
Hematocrit change (%) ... ... 1.00 (0.99–1.05) 1.00 1.00 (0.99–1.01) 0.87
White cell count change (x100) ... ... 1.01 (0.99–1.01) 0.22 1.00 (0.99–1.02) 0.28
Neutrophil count change (x100) ... ... 1.00 (0.96–1.03) 0.51 0.99 (0.95–1.01) 0.59
Platelet count change (x1000) ... ... 0.99 (0.96–1.00) 0.04 1.0 (0.99–1.00) 0.06
Therapy with liposomal amphotericin 7.03 (0.77–64.32) 0.08 6.35 (0.70–58.17) 0.10 5.97 (0.63–56.29) 0.12

Note. Model #1 includes clinical (but not laboratory findings). Model #2 includes laboratory (but not clinical findings). Model #3 includes both clinical and laboratory
findings. Significant results (P<0.05) are presented in boldface. HR, Hazard Ratio. CI, Confidence Interval.

Figure 2. Cox regression graphics for variables associated with relapse among 571 patients with visceral leishmaniasis successfully treated in inner Brazil.
Note. There were 38 relapses in the follow up period. Except for age groups (A), all other variables were independent predictors of relapse in the final multivariable models.

Discussion extensively documented (Cota et al., 2011, 2014; Gebreyohannes

et al., 2018). Factors such as previous relapses, absence of CD4+
In coherence with our findings, the outstanding negative count increase during therapy and lack of secondary prophylaxis
impact of HIV-coinfection on the outcome of VL has been have been blamed.
48 J.C. Simão et al. / International Journal of Infectious Diseases 95 (2020) 44–49
Since 2005, HIV-coinfected patients in our hospital were
treated with Liposomal Amphotericin 3–5 mg/kg for 5–7 days
and administered secondary prophylaxis with Liposomal Ampho-
tericin 3–5 mg/kg every 4 weeks (with no criteria for suspension of
prophylaxis). Only one out of 55 HIV-coinfected subjects in our
cohort was treated with Meglumine Antimoniate. Additional
criteria for the use of Liposomal Amphotericin included age under
10 years or over 50 years, pregnancy and immune suppression (de
Melo and Fortaleza, 2013). It is no surprise that apparently
increased relapse among those treated with Amphotericin was not
confirmed after adjusting for confounders.
Contrary to HIV-coinfection, data on relapse of VL in
comprehensive cohorts (or those including only the immunocom-
petent) were less studied. Relapse rates may be biased due to losses
of follow up, and were described as 1.4% in Bihar, India (Burza et al.,
2014) and 6.5% in South Sudan (Gorski et al., 2010). In Brazil, a
small open-label clinical trial found relapse in 7 out of 32 subjects
treated with Liposomal Amphotericin over a 5-year follow up
(Freire et al., 1997). A more robust clinical trial found overall 2,8%
relapse rate, but follow-up was limited to 180 days after therapy
(Kajaia et al., 2011). In short, extremes of age, persistence of
splenomegaly, shorter therapy regimens and low blood cell counts
have been identified as risk factors (Gorski et al., 2010; Kajaia et al.,
2011; Burza et al., 2014; Romero et al., 2017). Curiously, while some
authors identify delay in diagnosis as a predictor (Kajaia et al.,
2011), a robust Indian study found that subjects with a shorter
interval from symptoms to therapy were more likely to relapse
(Burza et al., 2014). Those findings should be interpreted in the
light of different epidemiological settings, etiological species and
therapeutic guidelines.
In our study, lower limb edema was an independent predictor of
VL relapse. We believe that this finding is a proxy measure for
malnourishment. It is worth noting that a previous study from our
group, which included body measures, bioimpedance and bio-
chemical assessment, found relevant evidence of malnourishment
in VL patients, as compared to healthy controls (Gatto et al., 2013).
Even though those measures could not be retrospectively assessed
in the present analysis, it is tempting to infer that the worse the
nutritional indicators, the greater the likelihood of relapse. This
opens an interesting venue for future prospective studies.
Among laboratory results, lower platelet counts (or alterna-
tively, lower platelet count improvement with therapy) indepen-
dently predicted relapse. It is reasonable to assume that these
variables measure either the intensity of the splenic sequestration
of blood cells or the bone marrow's ability to recover. Finally, the
diagnosis of secondary pneumonia during the first episode was a
significant risk factor for relapse. Not surprisingly, edema, low
platelet count and pneumonia were identified as predictors of
mortality in previous study enrolling Brazilian VL patients
(Druzian et al., 2015; Daher et al., 2017; Oliveira-Sena and
Werneck, 2019). Our findings point to an interesting similarity
among predictors for death and relapse. On the other hand,
frequently reported variables such as gender, age, duration of
symptoms, comorbidities other than HIV and – most importantly –
therapeutic choice did not impact relapses.
Our study is limited by the retrospective design and relatively
small geographical scope. Furthermore, there were changes in
therapeutic guidelines, with the introduction of Liposomal
Amphotericin B in 2005. Still, we could include a substantial
number of subjects without any loss to follow up. Also, a
comprehensive review and extensive analysis of predictors was
conducted.
A recent study in Brazil described relapse as an independent
risk factor for mortality (Druzian et al., 2015). This finding
emphasizes the relevance of identifying its predictors for guiding
the choice of appropriate therapy and for reinforcing the
importance of follow-up. Besides reaffirming the impact of HIV
coinfection on the outcome of VL, our study points to clinical and
laboratory findings that characterize patients who are more likely
to relapse. Multicenter studies and systematic reviews with
subgroup analyses may substantially increase our knowledge of
those predictors in the overall VL and HIV-seronegative subjects.
Certainly, subjects who are prone to relapses are eligible for a
closer follow-up, and possibly for novel therapeutic options, such
as a combination of drugs (Patole et al., 2014; Wasunna et al., 2016;
Mastroianni et al., 2018).
Funding
CMCBF received a researcher grant from the National Council
for Scientific and Technological Development (CNPq, Brazil; grant
# 310547/2018-0). The funding agency did not interfere with
design, data collection, analysis or writing of this manuscript.
Acknowledgments
C.M.C.B.F. and J.C.S. conceived this study. J.C.S. performed chart
reviews. C.V. and C.M.C.B.F. analyzed databases. All authors
contributed equally to writing this manuscript. Partial results of
this study were included in the PhD thesis of J.C.S., with C.M.C.B.F.
as advisor.
Ethics statement
This study was approved by the Committee for Ethics in Human
Research from Instituto Lauro de Souza Lima (City of Bauru, São
Paulo State, Brazil), approval number 37645014.1.0000.5475. Given
the nonconcurrent design, a waiver of application the informed
consent from study subjects was approved.
This study was approved by the Committee for Ethics in Human
Research from Instituto Lauro de Souza Lima (City of Bauru, São
Paulo State, Brazil), approval number 37645014.1.0000.5475. Given
the nonconcurrent design, a waiver of application the informed
consent from study subjects was approved.
Conflict of interest statement
All authors state that they have no conflict of interest regarding
this study.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.
ijid.2020.02.028.
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