Acute kidney injury in patients with Visceral Leishmaniasis in Northwest Ethiopia.

BACKGROUND: Visceral Leishmaniasis (VL) is a neglected tropical disease endemic to several countries including Ethiopia. Outside of Africa, kidney involvement in VL is frequent and associated with increased mortality. There is however limited data on acute kidney injury (AKI) in VL patients in East-Africa, particularly in areas with high rates of HIV co-infection. This study aims to determine the prevalence, characteristics and associated factors of AKI in VL patients in Northwest Ethiopia.
METHODS: A hospital based retrospective patient record analysis was conducted including patients treated for VL from January 2019 to December 2019 at the Leishmaniasis Research and Treatment Center (LRTC), Gondar, Ethiopia. Patients that were enrolled in ongoing clinical trials at the study site and those with significant incomplete data were excluded. Data was analyzed using SPSS version 20. P values were considered significant if < 0.05.
RESULTS: Among 352 VL patients treated at LRTC during the study period, 298 were included in the study. All were male patients except two; the median age was 23 years (IQR: 20-27). The overall prevalence of AKI among VL patients was 17.4% (confidence interval (CI): 13.6%-22.2%). Pre-renal azotemia (57%) and drug-induced AKI (50%) were the main etiologies of AKI at admission and post-admission respectively. Proteinuria and hematuria occurred in 85% and 42% of AKI patients respectively. Multivariate logistic regression revealed HIV co-infection (adjusted odds ratio (AOR): 6.01 95% CI: 1.99-18.27, p = 0.001) and other concomitant infections (AOR: 3.44 95% CI: 1.37-8.65, p = 0.009) to be independently associated with AKI.
CONCLUSION: AKI is a frequent complication in Ethiopian VL patients. Other renal manifestations included proteinuria, hematuria, and pyuria. HIV co-infection and other concomitant infections were significantly associated with AKI. Further studies are needed to quantify proteinuria and evaluate the influence of AKI on the treatment course, morbidity and mortality in VL patients.

Introduction

Leishmaniasis is a neglected parasitic tropical disease caused by various species of Leishmania. The diseases is endemic in more than 88 countries, with 350 million people at risk of infection [1]. Visceral Leishmaniasis (VL) is the most serious form of the disease with more than 95% of mortality if left untreated [2]. According to the 2017 WHO report, 94% of new cases of VL cases occurred in seven countries: Brazil, Ethiopia, India, Kenya, Somalia, South Sudan and Sudan [3]. The disease is endemic in numerous foci of the East African countries including Ethiopia, Kenya, South Sudan and Uganda [4]. VL in Ethiopia puts over 3.2 million people at risk, with up to 3000 new cases per year [1, 5]. The number of cases is associated with seasonal migration of labor force to endemic areas and HIV/AIDS [6].

Outside of East-Africa, renal involvement in VL has been found to be frequent and to be associated with increased mortality [7, 8]. Clinical features of renal involvement in VL are diverse including urinary abnormalities (proteinuria, hematuria, and pyuria), tubular dysfunction, glomerular dysfunction, and acute kidney injury (AKI) [9, 10]. AKI is a clinical syndrome characterized by a sudden decrease in glomerular filtration rate (GFR) sufficient to decrease the elimination of nitrogenous waste products and other uremic toxins [11]. Possible causes of development of AKI in VL patients are multifactorial which include the disease itself, drug toxicity, presence of associated infections and hemodynamic abnormalities [8, 12–16].

Different histological patterns of renal disease were reported in patients with VL including interstitial nephritis, proliferative glomerulonephritis, segmental necrotizing glomerulonephritis, and AA amyloid glomerular deposits [17-21].

Studies on renal abnormalities in East-Africa are very scarce. In a study done to assess the efficacy and safety of meglumine antimoniate, 9/54(30%) of Ethiopian VL patients developed abnormal renal function (defined as serum creatinine (SCr) >1.1mg/dl) [22]. Several studies on AKI have been done outside Africa where HIV prevalence is low and Sodium Stibogluconate (SSG), a potentially nephrotoxic drug, is the first line therapy. In Ethiopia, where SSG and Paromomycin (PM) combination is used as a first line treatment and where there is a high rate of HIV co-infection, there is scarcity of data on the actual prevalence of AKI in VL patients. The aim of this study was to determine the prevalence of AKI, characterize its manifestations and associated factors among VL patients in Northwest Ethiopia.

Methods

Study design and setting

This is a retrospective analysis of patients’ clinical data of VL patients treated at the Leishmaniasis Research and Treatment Center (LRTC) of the University of Gondar Hospital (UoGH) between January 2019 and December 2019. Data were retrieved from medical records over a period of two weeks, from February 3–15, 2020. UoGH is a tertiary-level teaching specialized public hospital located in Gondar town, Northwest Ethiopia. The hospital serves as the only referral hospital for patients coming from Northwest part of the country serving a catchment area for about 7 million populations. LRTC is located within UoGH and supported by the Drugs for Neglected Diseases Initiative (DNDi). LRTC has 24 inpatients beds and well equipped laboratory and pharmacy services. LRTC is one of the few clinical trial sites in Ethiopia and gives free diagnostic, treatment and follow-up service for VL patients presenting to the hospital. Diagnostic tests for VL including splenic and bone marrow aspiration and culture are routinely performed.

Close monitoring of patient’s response and safety are routine practice which includes monitoring of renal function tests, liver function tests and complete blood count (CBC) on weekly basis and when indicated. All patient clinical records including history, physical findings, laboratory tests, type of treatment and final outcome are documented on the medical charts of patients and kept in the center. The first line treatment for VL is SSG + PM and liposomal amphotericin B (L-AMB) is given for patients with critical illness, severe organ dysfunction and HIV co- infection.

Population, sample size, and sampling procedure

Medical charts of all patients admitted during the study period at LRTC with a diagnosis of VL were retrieved. Diagnosis of VL was made by either the presence of amastigotes on splenic or bone marrow aspirate—Giemsa-stain under light microscopy or a patient fulfilling WHO case definition for VL and positive rk-39 test if tissue aspiration was not done. Exclusion criteria were patients participating in ongoing clinical trials, if serum creatinine was not measured and if patient files could not be retrieved at LRTC chart room [23].

Sample size was calculated with an assumption of 50% of VL patients to have AKI at presentation or during treatment with 90% CI and 5% marginal error, yielding a sample size of 270. Accounting for 10% incomplete data, the total sample size was 297. Patients were included by retrograde consecutive sampling from December 2019 backward until the required sample size was achieved. Patients treated during this period were selected because this provides more recent data, representing the current situation in the study setting.

Variables and operational definitions

The outcome variable of interest was presence of AKI. The independent variables were age and sex, duration of illness, concomitant illness, cytopenia’s, HIV status, and treatment outcome of VL.

AKI definition and staging was as per the Kidney Disease Initiative Global Outcome (KDIGO) guideline [24]. AKI was defined as an increase in serum creatinine (SCr) by ≥0.3 mg/dl within 48 hours after admission; or an increase in SCr to ≥1.5 times baseline, which is known or presumed to have occurred within the prior 7 days. AKI was graded as stage 1 (SCr 1.5–1.9 times baseline or ≥0.3 mg/dl increase), stage 2 (SCr 2.0–2.9 times baseline) and stage 3 (SCr 3.0 times baseline or SCr to ≥4.0 mg/dl).

Resolved AKI was defined as normalization of SCr at discharge (to ≤1.1mg/dl) and non-resolved AKI was defined as not complete normalization of SCr. The AKI status was labelled as unknown if the SCr level was not known at the time of VL outcome assessment. Renal outcome was defined as good if the AKI resolved at discharge and bad if AKI had not resolved, was unknown or death in a patient with AKI not resolved at the time of death. The likely cause of AKI was based on clinical judgment of the treating physician.

VL clinical cure was defined as a patient showing improvement in signs and symptoms after standard treatment (fever resolution, hemoglobin increase, weight gain, and spleen size regression). VL parasitological cure was defined as improvement in signs and symptoms after standard treatment (fever resolution, hemoglobin increase, weight gain, and spleen size regression) and the absence of parasites in tissue aspirates. Treatment failure was defined as a positive test of cure (parasitological failure) and/or clinical signs/symptoms that persisted after completion standard treatment. Poor VL treatment outcomes included death, treatment failure or unknown outcome and good VL treatment outcome was defined as clinical or parasitological cure.

Data collection tool and analysis

Data were entered into Microsoft XL version 2016 and were exported to SPSS version 20 for statistical analysis. Data cleaning was performed before conducting descriptive analysis. Baseline characteristics were described using number and percentages. To examine the difference between independent variables among patients with or without AKI binary regression was performed. Both bivariate and multivariate logistic regression analyses were used to identify independently associated factors of AKI in VL patients. Those variables with a P-value <0.2 in the bivariate analysis were exported to multivariate analysis to control the possible effect of confounders. The Chi2 and Fisher exact test were used for binary/categorical variables and the Kruskal Wallis test was used for continuous variables. P values were considered significant if < 0.05. Missing data were not included in the analysis.

Ethical consideration

Ethical clearance and waiver of individual informed consent for this retrospective data analysis was obtained from the University of Gondar Institutional Review Board. Patient data confidentiality was respected at all levels from record review, chart retrieving and data analysis which was handled by the investigators.

Results

During the period between January 2019 and December 2019, a total of 1603 patients were screened and 352 were treated for VL at LRTC. Of these, 33 were included in ongoing clinical trials, two were excluded because of incomplete data and 19 patient files could not be retrieved. Finally, a total of 298 patients were included in the study for analysis (

Baseline demographic and clinical characteristics

All patients were male except two. The median age was 23 (IQR: 20–27) years. Almost all (97%) of the patients were in the age group between 15–45 years. The median duration of illness was one (IQR: 0.7–2) month. Patients were either referred from nearby health facilities or presented to UoG hospital by themselves. There were no patients who had received anti-leishmanial drugs before admission to the hospital.

More than half of the patients (53%) presented within one to three months after symptom onset. Around 70% of the patients had concomitant diagnoses like tuberculosis, intestinal parasitosis, pneumonia, otitis media and others. A total of 17 patients (6.1%) had VL-HIV co-infection, of which half of the cases (53%; 9/17) were on antiretroviral treatment at the time of VL diagnosis. The majority (95%) of patients had a first episode (primary) VL. Most patients (77.9%) were treated with a combination of SSG and PM for 17 days ().

Baseline laboratory characteristics

More than 95% of patients had anemia, leukopenia or thrombocytopenia. The majority (94.5%) of patients had low serum albumin; elevated transaminases was also a common finding. Renal manifestations like proteinuria, hematuria and pyuria were seen in 56.9%, 30.3% and 40.9% of VL patients respectively ().

Acute kidney injury in VL patients

Out of the 298 participants, a total of 52 (17.4%, confidence interval (CI): 13.6% - 22.2%) patients developed AKI. Of these, 30/298 (10%) had AKI at admission before treatment initiation and 22/268 (8.2%) patients developed AKI during the VL treatment course (11 during the first week, eight during the second week and two during the third week of treatment). Two third (66%) of patients had stage 1 AKI. Proteinuria occurred in 58% of patients with AKI while hematuria and pyuria were seen in 42% and 51% respectively ().

Factors associated with AKI

All baseline characteristics of VL patients were similar for patients with or without AKI except for the presence of HIV infection and concomitant infections, which tended to occur more commonly in patients with AKI. 85% of patients with AKI had concomitant infection while 67% of patients without AKI had concomitant infections (p = 0.0104). HIV co-infection was found in 17% and 4% of patients with and without AKI respectively (p = 0.0025) (). On bivariate analysis, factors significantly associated with AKI were duration of illness, presence of HIV co-infection and concomitant infections. On multivariate analysis, presence of HIV co-infection (adjusted odds ratio (AOR) = 6.01 95% CI: 1.99–18.27, p = 0.001) and other concomitant infections (AOR = 3.44 95% CI: 1.37–8.65, p = 0.009) were found to be associated with the development of AKI.

Drug induced AKI (post-admission AKI)

A total of 22 patients developed AKI after admission, yielding an incidence of 8.2% (22/268). The proportion of AKI per treatment allocation is shown in .

The main etiologies of AKI at admission and during the treatment course were dehydration and drug-induced AKI accounting for 57% and 50% of the patients respectively. Sixty seven percent (20/30) of the patients who had AKI at admission were treated with L-AMB. Out of the 22 patients who developed post-admission AKI, 11 (50%) patients had presumed drug-induced AKI due to SSG-based therapy and of these, six patients completed treatment using the same regimen. The remaining five were shifted to L-AMB due to worsening renal failure (data not shown).

The majority (40/52) of the patients with AKI had good renal outcome, with normalization of renal function. However, 12 patients had a bad renal outcome with two deaths, five AKI that had not resolved by the time of discharge or end of VL treatment. In the remaining five, the AKI status was unknown at the end of the VL treatment. One patient died of respiratory failure due to pneumonia and the other patient died of septic shock. In both cases, AKI had not resolved at the time of death ().

Discussion

This is one of the first studies from sub-Saharan Africa assessing the prevalence of AKI and other renal clinical manifestations in VL patients in Ethiopia. Renal involvement in VL has been reported before from outside of Africa in the form of urinary abnormalities, AKI and histological abnormalities in kidney biopsy [9, 25]. A total of 298 patients were included in the analysis and the spectrum of renal diseases included AKI (17.4%), proteinuria (56.9%), hematuria (30.3%) and pyuria (40.9%). The observed proportion of AKI (17.4%) was quite different from the assumed proportion (50%) used to calculate the sample size.

The proportion of AKI in patients with VL in our study was 17.4% (52/298). Our findings are similar to previous reports from India (15%) but an AKI prevalence of 26–37% has been reported from Brazil [25-27]. The reported incidence of AKI in VL varied from 4.2 to 45.9% [7, 10, 28]. The use of different criteria to define AKI and differences in etiological factors causing AKI may contribute to this variation in incidence of AKI in patients with VL [25].

In our study, proteinuria was found in 56.9% of VL patients using a semi-quantitative urine dipstick test. Microscopic hematuria and pyuria were observed in 30.3% and 40.9% of VL patients respectively but there was no difference in the occurrence of proteinuria and hematuria in patients with or without AKI (p > 0.05). This finding is consistent with previous studies. Abnormal urinary sediments (microscopic hematuria and proteinuria) in VL was observed by several investigators [18, 19]. A prospective study in Brazil in VL patients demonstrated hematuria, mild to moderate proteinuria, and increased urine leukocytes in over 50% of cases [29]. Another study from India, reported proteinuria ranging between 1.2–1.8gm/24 hrs in 15% of cases [25]. Proteinuria was also observed in patients with normal SCr and this is consistent with prior reports where proteinuria was detected in more than 40% of VL patients, even in those with normal SCr levels [30]. Urinary protein excretion in VL patients is usually in the range of <1 g/24 h [10]. However, as proteinuria was not measured quantitatively in our study, direct comparisons of our findings with these studies is not possible.

The most common cause of AKI at admission was pre-renal azotemia (fluid loss from diarrhea and vomiting, poor fluid intake). AKI in VL patients in previous reports was also mostly related to pre-renal factors [31]. Sepsis and drug induced AKI were the second most common causes of AKI in the current study. The first line of treatment for clinically stable HIV negative patients with VL in the study area is SSG + PM and most patients received this regimen while patients who are critically ill, having HIV-confection and significant renal and/or hepatic dysfunction received L-AMB.

The rate of post-admission AKI was 8.2% (22/268), of which eleven patients developed AKI due to presumed SSG + PM renal toxicity while only one patient had AKI due to presumed L-AMB renal toxicity (). However there was no statistically significant difference in the rate of post-admission AKI among patients treated with SSG-based and L-AMB based treatment (). Even though statistically not significant, patients who were treated with L-AMB regimen seems more likely to develop AKI compared with those treated with SSG-based regimen(OR = 2.17 CI:0.8–6.0). This is likely because patients who received L-AMB were either critically ill or have HIV co-infection and this will increase the risk of AKI independent of the anti-leishmanial treatment. Both SSG and PM are potentially nephrotoxic and AKI related to these drugs has been reported [31]. Previous clinical trials on safety and effectiveness of SSG and PM in East Africa have documented renal failure with fatality as severe adverse events [14]. L-AMB is generally very safe, however, increased serum urea and SCr due to amphotericin B have been reported [7, 9, 16]. One patient in our study developed AKI due to glomerulonephritis. This patient had renal failure with hypertension, active urine sediments, fluid overload and massive proteinuria of 6500mg/24hrs. Similar to this case, VL related glomerulonephritis has been reported [18, 32, 33].

In previous studies, several factors have been identified as risk factors for the development of AKI in VL patients including male gender, advanced age, jaundice, hypokalemia, leukopenia, use of conventional amphotericin B and secondary infections [8, 27]. 99.3% of the study participants in this study were male and this is consistent with prior reports of male predominance of 96–100% in the study area [22, 34]. This very significant male predominance could be due to presence of male seasonal daily laborers in the study area. In our study, a total of 17 patients (6.1%) had VL-HIV co-infection and HIV co-infection was an independent risk factor for the development of AKI. VL patients with HIV co-infection were 6 (AOR = 6.01 95% CI: 1.99–18.27, p = 0.001) times more likely to develop AKI compared to HIV negative VL patients VL is an important opportunistic disease in HIV-positive patients in Ethiopia. The Northwest part of Ethiopia, where this study was conducted, has the highest HIV-VL co-infection rate globally [34, 35]. The reasons for the increased incidence of AKI in HIV-VL co-infection could be multiple, including the occurrence of more severe VL, HIV per se, concomitant opportunistic infections or medication. There have been very few case reports of HIV-VL co-infected patients who developed renal complications. Reported renal manifestations in this group of patients included glomerular disease, nephrotic syndrome, AKI, and amyloidosis [13, 17, 20, 21].

In line with other studies, AKI also tended to occur more commonly in patients with concomitant secondary infections. Patients with concomitant secondary infections were 3.44 (AOR = 3.44 95% CI: 1.37–8.65, p = 0.009) times more likely to develop AKI than patients without secondary infections. Similarly to others, we also observed that AKI was reversible with correction of dehydration, treatment of concomitant bacterial infections and treatment of VL in the majority of cases [28, 31]. Additionally, mortality was low in this study. There were two fatalities in this study due to respiratory failure and refractory shock. Presence of AKI had likely contributed to the death in both patients. Routine monitoring of renal function is recommended to detect renal complications early on and guide patient management including fluid management and the choice of VL treatment.

There are several limitations to this study. Given the retrospective nature, there were some missing data, especially dipstick and urine microscopy were not done for all patients. Proteinuria was tested semi-quantitatively using urine dipstick; 24 hr urine protein excretion would have been more useful. Renal biopsy was not performed in those patients with features of glomerular disease, and hence it is difficult to characterize the nature of glomerular and tubular lesions. The etiology of AKI was based on the judgment of the treating physician and establishing the causality of AKI might be inaccurate.

Conclusion

In summary, renal disease occurred at the time of admission and during the course of treatment of VL patients. The spectrum of renal disease included AKI, proteinuria, hematuria, and pyuria. Presence of HIV co-infection and other secondary infections were independent risk factors for the development of AKI. The majority of the renal manifestations were generally of mild nature and reversible with treatment of VL, rehydration and treatment of bacterial co-infections without specific treatment. Further study is required to address the degree of proteinuria quantitatively, the nature of the glomerular and tubular lesions, the influence of AKI in the treatment course and its contribution for morbidity and mortality in patients with VL with and without HIV co-infection.

17 Feb 2021

PONE-D-21-00134

Acute Kidney Injury in patients with Visceral Leishmaniasis in Northwest Ethiopia

PLOS ONE

Dear Dr. Hailu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I enjoyed reading your manuscript and believe the reviewers have provided a number of constructive comments to improve clarity and help strengthen the manuscript.

Please submit your revised manuscript by Mar 26 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Andrea L. Conroy, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating in your ethics statement "As a retrospective study patient informed consent is waivered." Please clarify whether your ethics board specifically granted a waiver of informed consent. Please add this information to your manuscript and to the ethics statement in the online submission form.

3. Thank you for providing the date(s) when patient medical information was initially recorded. Please also include the date(s) on which your research team accessed/retrieved the medical records data used in your study.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"Our acknowledgement also goes to the Drugs for Neglected Disease Initiative (DNDI)

and University of Gondar (UoG) for supporting the Leishmaniasis Research and Treatment

Canter."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The authors received no specific funding for this work"

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

6. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

7. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://www.cell.com/heliyon/fulltext/S2405-8440(18)31817-6?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2405844018318176%3Fshowall%3Dtrue

- https://apps.who.int/iris/bitstream/handle/10665/311922/IC_Meet_Rep_2019_EN_20619.pdf

- https://cdn.intechopen.com/pdfs-wm/31093.pdf

- http://ispub.com/journal/the_internet_journal_of_tropical_medicine/volume_4_number_1_51/article/spectrum_of_renal_disease_in_visceral_leishmaniasis .html?

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for this study on AKI in Leishmaniasis; a topic which has limited data. Below are my comments in regard to the manuscript.

1) In the conclusion of the abstract, you report'....proteinuria, hematuria, pyuria and glomerulonephritis. How did you separate glomerulonephritis from hematuria and proteinuria.' Are these not presentations which made you diagnose those patients with glomerulonephritis? You need to review the statement and clarify how glomerulonephritis patients differed from those reported to have proteinuria and hematuria.

2) In the introduction you state'....Visceral Leishmaniasis (VL) is the most serious form of the disease with 100% mortality if left untreated.' May you provide a reference for the statement

3) In the introduction, you wrote '.....In Ethiopia, where SSG and PM combination is used as a first line treatment

and where there is a high rate of HIV'. However, PM abbreviation was not written in full. May you write in full on first use of the abbreviations

4) Under methods section you stated '....Patients included in ongoing clinical trials during this period, charts with significantly incomplete data or patient files that could not be retrieved at LRTC chart room were excluded. (22).' There is need to improve on the gramma in this sentence. It isn't clear in its current form.

5) When defining AKI, it is unclear what creatinine measurement was used to define baseline creatinine. This needs to be specified clearly

6) In the results you write...A total of 17 patients (6.1%) had VL-HIV co-infection, of which alf of the cases (53%; 9/17) were on antiretroviral. There is an error in this statement, it could be that the word 'alf' was to mean half

7) In table 1, specify which drug for VL is 'Other' since it was only used by one person

8) In general, there are a number of grammatical errors, there is need to improve the manuscript by ensuring that the gramma is improved.

9) Risk factors were based on a bivariate table 1. There was need for multivariate analysis to provide better results

10) It is unclear how AKI at admission was defined and how this definition differed from AKI during treatment.

There is also lack of clarity on how dehydration was determined and what was meant by nephrotoxicity; and what nephrotoxic drugs were being referred to or evaluated.

11) Table 3 and Table 4 provide etiology of AKI; from the study design it maybe hard to provide evidence of causality of AKI by these factors

12) In the conclusion section the researcher writes; 'In summary, renal disease occurred before VL diagnosis and during the course of treatment'. However, this may not be entirely based on the results since the study was evaluating patients admitted to hospital and not before diagnosis. The study doesn't evaluate patient before VL diagnosis but provides data on patients who had been managed for VL at the VL treatment centre. There is need to make more clarification on this and the conclusion from the study.

In addition, the researcher states in the conclusion that the renal manisfestations were mild in nature and reversible; however, there was stage 3 AKI and the research never provided recovery data to support the statement of reversibility

Reviewer #2: The authors are to be congratulated on this interesting report of AKI in VL from Ethiopia.

The causes of AKI at admission (present in 10% of patients) included dehydration. HIV and other co-infections were risk factors for AKI (interesting).

AKI also developed in 22 patients who did not have AKI at admission, which could be related to medications.

This manuscript warrants eventual publication, but I recommend some revisions to improve the clarity of the findings reported. As outlined below, the reporting of possible drug-associated AKI is inadequate and possibly even misleading.

Major revision: improved reporting of possible drug-associated AKI

AKI is multi-factorial in this patient cohort.

The authors have done well to separate AKI at admission from AKI during treatment to tease out the effect of medications, for example. Can the authors please confirm (and report in the revised manuscript methods and/or results), whether patients referred to Gondar Hospital had any prior treatment for their VL or other conditions at other centres? Prior treatment with L-AMB or pentavalent antimonials or aminoglycosides/vancomycin/ other nephrotoxic antibiotics. It’s not explicitly stated whether the patients were treatment experienced (potential exposure to nephrotoxins) or treatment naïve. If treatment naïve, this is the “natural history” of AKI in VL as opposed to iatrogenic. As a referral centre, it is possible that patients had been previously hospitalized and treated elsewhere with or without an appropriate diagnosis of VL, for example.

The development of incident AKI after admission in 22 patients may be related to medications (SSG or L-AMB). I strongly recommend that the rate of post-admission AKI be expressed as the proportion of those without AKI at admission who subsequently develop AKI (authors are using the wrong denominator when they divide by the whole cohort n=298):

I believe there should be 268 patients without AKI at admission (298-30):

22/268 = 8.2% (denominator is those without AKI at admission).

I recommend that the rate be compared in those treated with L-AMB and those treated with SSG:

Of the 268 patients without AKI at admission, how many were treated with L-AMB, how many with SSG?

What was the rate of AKI in the SSG group? (excluding patients with AKI at admission)

What was the rate of AKI in the L-AMB group? (excluding patients with AKI at admission)

Could consider a statistical comparison.

If these data can be deduced from the tables 1 and 4, I get the following (excluding patients with AKI at admission):

Drug no AKI post-admission AKI

SSG 204 19

L-AMB 38 3

Total: 242 22 Overall total: 264 (approx. 268, excluding AKI at admission)

Rate of AKI in SSG group: 19/223 = 8.5%

Rate of AKI in L-AMB exposed: 3/41 = 7.3%

OR = 1.16, p-value >0.99

This level of detail is essential to understand the effects of the medications on incident AKI after treatment initiation.

The current reporting is not sufficiently detailed and even misleading:

“Most (67%; 20/30) patients who had AKI at admission were treated with AmBisomeTM whereas 86% (19/22) of patients who develop AKI during VL treatment course were on SSG +PM”

After initial read, I though the SSG was associated with AKI, but a more careful review of the data suggests to me there is no evidence of higher rates of incident AKI in the SSG-treated patients.

This is partially addressed in Table 4. However, with respect to drug treatment, it would be helpful to add to table 4 those patients who did not have AKI at admission or during treatment (n=246, control group without AKI at admission, who did not develop AKI subsequently)– which drugs received and whether the drugs were switched. This could be included as a third column in Table 4 (I realize there wouldn’t be entries for “etiology of AKI” in this group!). This is an essential comparator group to make sense of the treatment numbers.

In summary, the reporting of AKI after admission according to treatment is not sufficiently detailed (Major revision necessary since this is a central mechanism of the AKI in this study).

Minor revisions:

Abstract:

17.4% (52/298) VL patients had AKI: recommend giving the 95% confidence interval on this proportion, since it’s the primary objective of the study.

AmBisome: liposomal amphotericin B. Please use generic rather than trade name

Introduction:

Glucantime: meglumine antimoniate. please use generic rather than trade name. (Introduction, 3rd paragraph)

PM: paromomycin? Please spell out the name with abbreviation when first mentioned (Introduction, 3rd paragraph)

Methods:

“all patients admitted during the study period at LRTC with confirmed diagnosis of VL” – should state the way the diagnosis was confirmed (e.g., confirmed by the presence of amastigotes on splenic or bone marrow aspirate - Giemsa-stain, light microscopy…) Please explicitly state if serology, PCR, and culture were not used for diagnosis.

Sample size calculation:

First, the authors are to be congratulated on including a sample size calculation for this observational study (frequently omitted).

I note that the authors used the alpha=90% level of significance for this calculation (z-score[0.05]=1.64):

n=(p)*(1-p)*(1.64/d)^2 = (0.5)*(1-0.5)*(1.64/0.05)^2 = 270

whereas alpha=95%CI is more standard (z-score[0.025]=1.96):

n=(p)*(1-p)*(1.96/d)^2 = (0.5)*(1-0.5)*(1.96/0.05)^2 = 378

In the end, the estimate of the proportion (50%) is quite different from the observed proportion (17%) and the confidence interval on the proportion is still within +/-5%

Overall, I think the sample size is adequate, but the assumptions of the sample size calculation are different from convention and from the observed proportion. A brief comment in the discussion on the adequacy of the sample size and differences between a priori assumed rate and observed rate may be considered.

The sampling strategy for the chart review is also clear and I congratulate the authors for describing this.

Results.

All but two of the charts reviewed were of male patients?

This is surprising. Although there is a male predominance in VL, it is far from an exclusively male disease. (e.g., male/female ratio was 1.40 in PLOS NTD, Male predominance in reported Visceral Leishmaniasis cases: Nature or nurture? A comparison of population-based with health facility-reported data.)

The ratio in this study would be 296:2 !

I think this requires an explanation why female patients were not included (not likely chance alone).

Risk factors for AKI (Co-infection and HIV): please give odds ratio and 95%CI for the association between HIV and AKI, likewise for co-infections and AKI.

Editorial/language revisions:

clinician judgment of the treating physician: clinical judgment of the treating physician (Methods, Variables and operational definitions, paragraph 2)

Patient’s data confidentially were respected: patient data confidentiality was respected (Methods, ethical considerations)

“The main etiology of AKI at admission and during treatment course were dehydration and nephrotoxicity” – "nephrotoxicity" is not really an etiology (too general a term) – perhaps reword: medication-induced tubular injury or glomerular injury, for example. (Results)

Table 2: AST elevation. LN (missing U for ULN)

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Michael Hawkes

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Apr 2021

1. Response to the academic editor’s comments

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reply: Thank you for the comments. We have edited both the authors’ affiliation and the main manuscript body as per the PLOS ONE style requirement.

2. Thank you for stating in your ethics statement "As a retrospective study patient informed consent is waivered." Please clarify whether your ethics board specifically granted a waiver of informed consent. Please add this information to your manuscript and to the ethics statement in the online submission form.

Reply: Yes, the ethics board has granted a waiver of informed consent and we have added this information to the manuscript and in the online submission form. Line #175-176

3. Thank you for providing the date(s) when patient medical information was initially recorded. Please also include the date(s) on which your research team accessed/retrieved the medical records data used in your study.

Reply: This is a relevant remark. Data was retrieved from the medical records over a period of 2 weeks (Feb 3-15, 2020). We have included this in the “methods” part, line # 107-108.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"Our acknowledgement also goes to the Drugs for Neglected Disease Initiative (DNDI)

and University of Gondar (UoG) for supporting the Leishmaniasis Research and Treatment

Canter."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The authors received no specific funding for this work"

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Reply: We haven’t got any specific fund for this work. We retrieved the data from LRTC. This center is funded by DNDi. That is why we put DNDI on the acknowledgment part.

5. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

Reply: The ethical statement is moved to the methods part and removed from the declaration section. Line #175-179

6. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

Reply: All authors’ affiliation is to University of Gondar where the work has been done EXCEPT for Johan van Grienvsen whose affiliation is to ITM, Belgium. Even though he is not a member of the University of Gondar, his contribution to this manuscript is significant to be a co-author.

7. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://www.cell.com/heliyon/fulltext/S2405-8440(18)31817-6?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2405844018318176%3Fshowall%3Dtrue

- https://apps.who.int/iris/bitstream/handle/10665/311922/IC_Meet_Rep_2019_EN_20619.pdf

- https://cdn.intechopen.com/pdfs-wm/31093.pdf

- http://ispub.com/journal/the_internet_journal_of_tropical_medicine/volume_4_number_1_51/article/spectrum_of_renal_disease_in_visceral_leishmaniasis .html?

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Reply: Thank you. We have rephrased and used our own words to avoid overlapping texts with the above mentioned 4 previous publications that we cited. Line #71, 72, 77, 85

2. Answer to the reviewer’s comments

Reviewers' comments:

Reviewer #1: Thank you for this study on AKI in Leishmaniasis; a topic which has limited data. Below are my comments in regard to the manuscript.

Reply: Thanks for the positive feedback

1) In the conclusion of the abstract, you report'....proteinuria, hematuria, pyuria and glomerulonephritis. How did you separate glomerulonephritis from hematuria and proteinuria.' Are these not presentations which made you diagnose those patients with glomerulonephritis? You need to review the statement and clarify how glomerulonephritis patients differed from those reported to have proteinuria and hematuria.

Reply: The question is indeed relevant. Yes proteinuria and hematuria are the cardinal features of glomerulonephritis. In this study, all but one of the patients had either proteinuria or hematuria without having other clinical features of glomerulonephritis (edema, hypertension and renal insufficiency). The causes of these urinary findings could be fever, sepsis or glomerulonephritis (but we haven’t performed biopsy to confirm this). There was only one patient who had anasarca, massive proteinuria, hypertension and renal failure fulfilling all the clinical criteria for glomerulonephritis. We have removed glomerulonephritis from the conclusion part of the statement (as there is just one case). Line # 54 and 401

2) In the introduction you state'....Visceral Leishmaniasis (VL) is the most serious form of the disease with 100% mortality if left untreated.' May you provide a reference for the statement

Reply: While searching prognosis of untreated VL, we found it is around 95% fatality (WHO fact sheet). We have adjusted this statement accordingly and included the reference. Line# 75

3) In the introduction, you wrote '.....In Ethiopia, where SSG and PM combination is used as a first line treatment

and where there is a high rate of HIV'. However, PM abbreviation was not written in full. May you write in full on first use of the abbreviations

Reply: Thanks, corrected accordingly. Line #99

4) Under methods section you stated '....Patients included in ongoing clinical trials during this period, charts with significantly incomplete data or patient files that could not be retrieved at LRTC chart room were excluded. (22).' There is need to improve on the gramma in this sentence. It isn't clear in its current form.

Reply: we have edited the statement like this” Exclusion criteria were patients participating in ongoing clinical trials, if serum creatinine was not measured and if patient files could not be retrieved at the LRTC chart room” Line# 128-132.

5) When defining AKI, it is unclear what creatinine measurement was used to define baseline creatinine. This needs to be specified clearly

Reply: We used the KDIGO definition as it is. For patients who developed AKI after admission, we use the initial creatinine determined in the hospital as the baseline creatinine. But for those who had elevated creatinine from the outset (at admission), and if it is presumed to occur within the last 7 days, we take it as AKI (that is why we mentioned “Known or presumed” to occur within the past 7 days on the definition part. This is true also in clinical practice that most patients with AKI do not have baseline creatinine before admission, but if they present with risk factors that can cause AKI, the elevated creatinine will be assumed to be of new onset.

6) In the results you write...A total of 17 patients (6.1%) had VL-HIV co-infection, of which alf of the cases (53%; 9/17) were on antiretroviral. There is an error in this statement, it could be that the word 'alf' was to mean half

Reply: yes, this is typo (we mean”half”) - corrected. Line # 194

7) In table 1, specify which drug for VL is 'Other' since it was only used by one person

Reply: This patient switched to more than two regimens. Hence it was difficult to put a single treatment allocation. We put a foot note under table 1 to describe this.

8) In general, there are a number of grammatical errors, there is need to improve the manuscript by ensuring that the gramma is improved.

Reply: Thanks, we have tried to improve the grammatical errors in the revised manuscript.

9) Risk factors were based on a bivariate table 1. There was need for multivariate analysis to provide better results

Reply: This is a valid comment. We run a multivariate analysis and we found that HIV status and concomitant infections were factors that were significantly associated with development of AKI. We have provided this information on the result section. Line # 245-249. The abstract is revised according to this finding. Line# 48-50

10) It is unclear how AKI at admission was defined and how this definition differed from AKI during treatment.

Reply: As we tried to explain above, AKI at admission was an elevated serum creatinine that was presumed to occur recently (within 7 days) - see definition. While AKI after admission was an elevation in creatinine level from the baseline creatinine that was determined at admission.

There is also lack of clarity on how dehydration was determined and what was meant by nephrotoxicity; and what nephrotoxic drugs were being referred to or evaluated.

Reply: These possible causes of AKI were assigned by the treating physician (pre-renal/dehydration- those patients having AKI and, history of fluid loss from diarrhea, vomiting with low blood pressure), drug induced AKI is defined if a patient develop AKI that is presumed due to the VL treatment drugs (excluding sepsis and pre-renal causes). The drugs referred here are SSG, PM and AmBisome (Table 3). We changed the term nephrotoxicity to drug induced AKI (as nephrotoxicity is a very broad term)

11) Table 3 and Table 4 provide etiology of AKI; from the study design it maybe hard to provide evidence of causality of AKI by these factors

Reply: We understand this concern. It may be difficult to provide of causality with the method we used. We used the treating physician’s judgment as a cause of the AKI (mentioned in the operational definitions). This might be inaccurate. In fact AKI etiology is usually obtained clinically (from history, physical examination and lab data) in routine clinical practice. Biopsy is rarely indicated to find etiology of AKI (glomerulonephritis and interstitial nephritis). Nephrotoxicity (considering the time relationship) and pre-renal AKI are usually clinically diagnosed. But we acknowledge your concern and we mentioned this in the limitation part of the revised manuscript. Line # 404-405

We have also amended table 4 as per a comment from the other reviewer.

12) In the conclusion section the researcher writes; 'In summary, renal disease occurred before VL diagnosis and during the course of treatment'. However, this may not be entirely based on the results since the study was evaluating patients admitted to hospital and not before diagnosis. The study doesn't evaluate patient before VL diagnosis but provides data on patients who had been managed for VL at the VL treatment centre. There is need to make more clarification on this and the conclusion from the study.

Reply: This point is indeed relevant. Yes we agree that, we have seen the patients with VL diagnosis at admission and during follow up. However, for the vast majority of the patients, diagnosis of VL was made at the center after self/health facility referral. We have corrected this statement accordingly as” In summary, renal disease occurred at the time of admission and during the course of treatment of VL patients”. Line # 407-408

In addition, the researcher states in the conclusion that the renal manisfestations were mild in nature and reversible; however, there was stage 3 AKI and the research never provided recovery data to support the statement of reversibility

Reply: 77% of all the AKI have resolved (Table 3). Yes, there were 8 patients with stage 3 AKI, but we didn’t follow outcome specifically for the stage 3. We corrected this statement as ”The majority of the renal manifestations were generally of mild nature and reversible with treatment of VL, rehydration and treatment of bacterial co-infections without specific treatment in the majority of cases” Line # 412

Reviewer #2: The authors are to be congratulated on this interesting report of AKI in VL from Ethiopia.

The causes of AKI at admission (present in 10% of patients) included dehydration. HIV and other co-infections were risk factors for AKI (interesting).

AKI also developed in 22 patients who did not have AKI at admission, which could be related to medications.

This manuscript warrants eventual publication, but I recommend some revisions to improve the clarity of the findings reported. As outlined below, the reporting of possible drug-associated AKI is inadequate and possibly even misleading.

Reply: Thanks for the positive feedback

Major revision: improved reporting of possible drug-associated AKI

AKI is multi-factorial in this patient cohort.

The authors have done well to separate AKI at admission from AKI during treatment to tease out the effect of medications, for example. Can the authors please confirm (and report in the revised manuscript methods and/or results), whether patients referred to Gondar Hospital had any prior treatment for their VL or other conditions at other centres? Prior treatment with L-AMB or pentavalent antimonials or aminoglycosides/vancomycin/ other nephrotoxic antibiotics. It’s not explicitly stated whether the patients were treatment experienced (potential exposure to nephrotoxins) or treatment naïve. If treatment naïve, this is the “natural history” of AKI in VL as opposed to iatrogenic. As a referral centre, it is possible that patients had been previously hospitalized and treated elsewhere with or without an appropriate diagnosis of VL, for example.

Reply: These points are indeed relevant. Regarding the AKI at admission, it is likely due to the natural history. All patients were treatment naïve. Prior treatment with anti-leishmanial drug or other nephrotoxic medications was not given. These are patients who visited the hospital by self-referral or referred from health centers in the vicinity. We have mentioned this in the result section (baseline demographic and clinical characteristics). Line # 188-190

The development of incident AKI after admission in 22 patients may be related to medications (SSG or L-AMB). I strongly recommend that the rate of post-admission AKI be expressed as the proportion of those without AKI at admission who subsequently develop AKI (authors are using the wrong denominator when they divide by the whole cohort n=298):

I believe there should be 268 patients without AKI at admission (298-30):

22/268 = 8.2% (denominator is those without AKI at admission).

Reply: This is absolutely true. We have corrected the rate of post-admission AKI using the 268 patients as a denominator. Line # 234-235.

I recommend that the rate be compared in those treated with L-AMB and those treated with SSG:

Of the 268 patients without AKI at admission, how many were treated with L-AMB, how many with SSG?

What was the rate of AKI in the SSG group? (excluding patients with AKI at admission)

What was the rate of AKI in the L-AMB group? (excluding patients with AKI at admission)

Could consider a statistical comparison.

If these data can be deduced from the tables 1 and 4, I get the following (excluding patients with AKI at admission):

Drug no AKI post-admission AKI

SSG 204 19

L-AMB 38 3

Total: 242 22 Overall total: 264 (approx. 268, excluding AKI at admission)

Rate of AKI in SSG group: 19/223 = 8.5%

Rate of AKI in L-AMB exposed: 3/41 = 7.3%

OR = 1.16, p-value >0.99

This level of detail is essential to understand the effects of the medications on incident AKI after treatment initiation.

The current reporting is not sufficiently detailed and even misleading:

“Most (67%; 20/30) patients who had AKI at admission were treated with AmBisomeTM whereas 86% (19/22) of patients who develop AKI during VL treatment course were on SSG +PM”

After initial read, I though the SSG was associated with AKI, but a more careful review of the data suggests to me there is no evidence of higher rates of incident AKI in the SSG-treated patients.

This is partially addressed in Table 4. However, with respect to drug treatment, it would be helpful to add to table 4 those patients who did not have AKI at admission or during treatment (n=246, control group without AKI at admission, who did not develop AKI subsequently)– which drugs received and whether the drugs were switched. This could be included as a third column in Table 4 (I realize there wouldn’t be entries for “etiology of AKI” in this group!). This is an essential comparator group to make sense of the treatment numbers.

In summary, the reporting of AKI after admission according to treatment is not sufficiently detailed (Major revision necessary since this is a central mechanism of the AKI in this study).

Reply: All the raised points are indeed relevant. Thank you for the detailed explanation on how to do the analysis for the drug induced AKI. We just took the frequency of patients who took SSG or L-AMB only from those who develop AKI, and it ends up with a wrong conclusion. According to your comment, we ran the data for the 268 patients, to look at AKI development per treatment arm (we see exposure-SSG or L-AMB) and looked for the rate of outcome (AKI) per treatment allocation. We have reported this in the new Table 4 (we totally changed table 4). Table 3 is also slightly modified.

As you mentioned above, there is no evidence supporting higher rates of AKI in SSG than L-AMB. Rather the trend seems patients on L-AMB likely to develop AKI than those on SSG based. This is probably because patients who are assigned to L-AMB from the beginning are those who are either critically ill or have HIV co-infection. Such patients are at risk of developing AKI and they were put on L-AMB for fear of SSG toxicity. We mentioned this in the discussion part. Line # 357-363

Minor revisions:

Abstract:

17.4% (52/298) VL patients had AKI: recommend giving the 95% confidence interval on this proportion, since it’s the primary objective of the study.

Reply: A relevant remark. We have included 95% CI. Line # 39-40

AmBisome: liposomal amphotericin B. Please use generic rather than trade name

Reply: we have corrected it accordingly

Introduction:

Glucantime: meglumine antimoniate. please use generic rather than trade name. (Introduction, 3rd paragraph)

Reply: Corrected

PM: paromomycin? Please spell out the name with abbreviation when first mentioned (Introduction, 3rd paragraph)

Reply: corrected . Line # 99

Methods:

“all patients admitted during the study period at LRTC with confirmed diagnosis of VL” – should state the way the diagnosis was confirmed (e.g., confirmed by the presence of amastigotes on splenic or bone marrow aspirate - Giemsa-stain, light microscopy…) Please explicitly state if serology, PCR, and culture were not used for diagnosis.

Reply: Majority of patients have a parasitological diagnosis of VL. However, there were patients diagnosed with clinical and serologic criteria. We have adjusted the statement like” Medical charts of all patients admitted during the study period at LRTC with diagnosis of VL were retrieved. Diagnosis of VL was made by either the presence of amastigotes on splenic or bone marrow aspirate - Giemsa-stain under light microscopy or a patient fulfilling WHO case definition for VL and positive rK-39 test if tissue aspiration was not done” Line # 126-128

Sample size calculation:

First, the authors are to be congratulated on including a sample size calculation for this observational study (frequently omitted).

I note that the authors used the alpha=90% level of significance for this calculation (z-score[0.05]=1.64):

n=(p)*(1-p)*(1.64/d)^2 = (0.5)*(1-0.5)*(1.64/0.05)^2 = 270

whereas alpha=95%CI is more standard (z-score[0.025]=1.96):

n=(p)*(1-p)*(1.96/d)^2 = (0.5)*(1-0.5)*(1.96/0.05)^2 = 378

In the end, the estimate of the proportion (50%) is quite different from the observed proportion (17%) and the confidence interval on the proportion is still within +/-5%

Overall, I think the sample size is adequate, but the assumptions of the sample size calculation are different from convention and from the observed proportion. A brief comment in the discussion on the adequacy of the sample size and differences between a priori assumed rate and observed rate may be considered.

Reply: we have included a brief comment on this under the first paragraph of the discussion part. Line # 322-323

The sampling strategy for the chart review is also clear and I congratulate the authors for describing this.

Reply: Thank you for your positive feedback

Results.

All but two of the charts reviewed were of male patients?

This is surprising. Although there is a male predominance in VL, it is far from an exclusively male disease. (e.g., male/female ratio was 1.40 in PLOS NTD, Male predominance in reported Visceral Leishmaniasis cases: Nature or nurture? A comparison of population-based with health facility-reported data.)

The ratio in this study would be 296:2 !

I think this requires an explanation why female patients were not included (not likely chance alone).

Reply: This is an interesting point. Females were not excluded from the study. Significant male predominance of VL cases is the usual scenario, here in northwest Ethiopia. Our previous published reports also showed 97-100% of VL cases were male. Most of the VL cases in the northwest Ethiopia are seasonal migrant laborers who came from different parts of Ethiopia to harvest cash crops (and almost all are males). We discussed this briefly on the discussion part and put the references. Line # 373-376

Risk factors for AKI (Co-infection and HIV): please give odds ratio and 95%CI for the association between HIV and AKI, likewise for co-infections and AKI.

Reply: We run a multivariate regression analysis and we found that HIV status and other concomitant infections were significantly associated with the development of AKI. We provide the adjusted odds ratio and 95%. Line # 241-246. We have modified the result section of the abstract accordingly.

Editorial/language revisions:

clinician judgment of the treating physician: clinical judgment of the treating physician(Methods, Variables and operational definitions, paragraph 2)

Reply: corrected. Line # 154

Patient’s data confidentially were respected: patient data confidentiality was respected (Methods, ethical considerations)

Reply: sentence adjusted. Line # 178

“The main etiology of AKI at admission and during treatment course were dehydration and nephrotoxicity” – "nephrotoxicity" is not really an etiology (too general a term) – perhaps reword: medication-induced tubular injury or glomerular injury, for example. (Results)

Reply: we changed to” drug –induced AKI”- which is usually tubular injury

Table 2: AST elevation. LN (missing U for ULN) → corrected

Submitted filename: Response to reviewers.docx

Click here for additional data file.

17 May 2021

Acute Kidney Injury in patients with Visceral Leishmaniasis in Northwest Ethiopia

PONE-D-21-00134R1

Dear Dr. Hailu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Andrea L. Conroy, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Thank you for thoroughly addressing my comments. All revisions have been done to my satisfaction. The manuscript is very interesting!

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Michael Hawkes

31 May 2021

PONE-D-21-00134R1

Acute Kidney Injury in patients with Visceral Leishmaniasis in Northwest Ethiopia

Dear Dr. Hailu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Andrea L. Conroy

Academic Editor

PLOS ONE

Table 1

Baseline demographic and clinical characteristics of VL patients at LRTC between January 2019 –December 2019, Gondar, Ethiopia, N = 298.

Variables	Total N (%)	AKI	No AKI	P value
		N (%)	N (%)
Age, median (IQR); (n = 298)	23 (20–27)	24 (21–27)	23 (20–26)	0.0997
< 15 years	1 (0.3)	0 (0)	1 (0)	1
15–45 years	290 (97.3)	48 (92)	242 (98)
> 45 years	7 (2.3)	4 (8)	3 (1)
Gender (n = 298)
Male	296 (99.3)	52 (100)	244 (99)	1
Female	2 (0.7)	0 (0)	2 (1)
Duration of illness (n = 298), median (IQR)	1 (0.7–2)	1 (0.5–2)	1.9 (0.7–2)	0.1493
< 1 month	94 (31.5)	21 (40)	73 (30)	0.0744
1–3 months	159 (53.4)	28 (54)	131 (53)
> 3 months	45 (15.1)	3 (6)	42 (17)
Concomitant Infections (n = 298)				0.0104
No	90 (30.2)	8 (15)	82 (33)
Yes	208 (69.8)	44 (85)	164 (67)
Tuberculosis	65 (31.1)	13 (30)	52 (32)	0.8
Intestinal parasitosis	62 (29.7)	11 (25)	51 (31)	0.44
Otitis media	4 (1.9)	2 (5)	2 (1)	0.1958
Pneumonia	9 (4.3)	3 (7)	6 (4)	0.4014
Others	69 (33)	15 (7)	54 (33)	0.8643
VL occurrence (n = 294)				0.3135
First episode	279 (94.9)	48 (92)	231 (95)
Relapse	15 (5.1)	4 (8)	11 (5)
HIV status (n = 275)				0.0025
Negative	261 (93.9)	39 (83)	226 (96)
Positive	17 (6.1)	8 (17)	9 (4)
Type of VL treatment (n = 294)
SSG + PM	229 (77.9)	25 (50)	204 (84)	<0.001
SSG alone	2 (0.7)	0 (0)	2 (1)	1
L-AMB	58 (19.7)	22 (44)	36 (15)	<0.001
L-AMB + Miltefosine	4 (1.4)	2 (4)	2 (1)	0.1355
Other *	1 (0.3)	1 (2)	0 (0)	0.1701
VL treatment outcome (n = 295)				0.2865
Good	280 (94.9)	45 (92)	235 (96)	0.2865
Poor	15 (5.1)	4 (8)	11 (4)

AKI: acute kidney injury, IQR: Inter Quartile Range, VL: Visceral Leishmaniasis, HIV: Human Immunodeficiency Virus, SSG: Sodium stibogluconate, PM: Paromomycin, L-AMB: Liposomal amphotericin B.

* This patient switched to more than two regimens.

Table 2

Baseline laboratory characteristics of VL patients at LRTC between January 2019 –December 2019, Gondar, Ethiopia, N = 298.

Variables	Total N (%)	AKI	No AKI	P value
		N (%)	N (%)
WBC, median (IQR) (n = 278)	1.4 (1.1–2.0)	1.42 (1.09–1.9)	1.4 (1.1–2.0)	0.999
Leukopeniaa	266 (95.7)	48 (92)	238 (97)	0.2336
Normal, n (%)	12 (4.3)	4 (8)	8 (3)
Hemoglobin median IQR (n = 298)	8.3 (6.7–10.8)	7.9(6.6–9.3)	8.5 (6.68–9.6)	0.2395
Anemiab	298 (100)	52 (100)	246 (100)	1
No anemia	0	0	0
Platelet count, median (IQR) (n = 294)	52 (31–85)	50 (29.5–74.0)	51 (31–84)	0.5
Mild thrombocytopeniac	38 (12.9)	4 (8)	34 (14)	0.2545
Moderate thrombocytopenia	112 (38.1)	21 (42)	91 (37)	0.5326
Severe thrombocytopenia	132 (44.9)	22 (44)	110 (45)	0.8886
Normal platelets	12 (4.1)	3 (6)	9 (4)	0.5347
Proteinuriad (n = 225)				0.8908
Yes	128 (56.9)	22 (58)	106 (57)
No	97 (43.1)	16 (42)	81(43)
Hematuriae (n = 221)				0.0823
Yes	67(30.3)	16 (42)	51(28)
No	154 (69.7)	22 (58)	132 (72)
Pyuriaf (n = 220)				0.1566
Yes	90 (40.9)	19 (51)	71 (39)
No	130 (59.1)	18 (49)	112 (61)
Hypoalbuminemiag (n = 275)				1
Yes	261 (94.9)	47 (96)	214 (95)
No	14 (5.1)	2 (4)	12 (5)
Hyperbilurubinemiah (n = 171)				0.3462
Yes	60 (35.1)	12 (43)	48 (34)
No	111 (64.9)	16 (57)	95 (66)
T-Bil median/IQR (n = 171)	1.4 (.9–3.62)	0.85 (.05–2.48)	0.85 (.5–1.3)	0.5476
D-Bil median/IQR (n = 33)	1.2 (.4–2.3)	1.9 (1.6-.4.5)	1.1 (.3–2.1)	0.1452
Creatinine median/IQR (n = 298)	0.9 (0.7–1)	1.3 (.9–1.7)	0.8 (.7-.9)	0.7848
AST elevation (n = 291)				0.4981
Normal	48 (16.1)	12 (23)	36 (15)
1–3 X ULN	132 (44.3)	22 (42)	110 (45)
3–5 X ULN	70 (23.5)	11 (21)	59 (24)
>5 ULN	48 (16.1)	7 (13)	41 (17)
ALT elevation (n = 267)
Normal	156 (52.3)	31 (60)	125 (51)	0.6190
1–3 X ULN	122 (40.9)	18 (35)	104 (42)	0.8837
3–5 X ULN	13 (4.4)	3 (6)	10 (4)	0.6658
>5 X ULN	7 (2.3)	0/0	7 (3)	1

AKI: acute kidney injury, IQR: Inter Quartile Range, WBC: White blood cells, BilT: Total bilirubin, BilD: Direct bilirubin, AST: Aspartate Aminotransferase, ALT: Alanine Aminotransferase, ULN: Upper Limit of Normal.

a: leukopenia is defined as WBC count <4000.

b: anemia is defined as Hgb<12mg/dl for female and < 13mg/dl for male.

c: thrombocytopenia defined as mild when platelet count s between 100, 000 and 150,000, moderate as a count of 50000 and 100000 and severe if count is less than 50000.

d. proteinuria is defined as urine dipstick for protein ≥ 1+ or 24 hr protein >150mg/day.

e. hematuria is defined as ≥3RBCs/High power field (HPF) on urine microscopy.

f. pyuria is defined as ≥ 5WBC/HPF on urine microscopy.

g: hypoalbuminemia is defined as serum albumin value less than 3.5gm/dl.

h: hyperbilirubinemia is defined as a total bilirubin value increased above the upper normal value, 1.2mg/dl.

Table 3

Clinical characteristics of VL patients with AKI at LRTC between January 2019 –December 2019, Gondar, Ethiopia, N = 52.

Etiology of AKI (n = 52)	N	%
Pre-renal (dehydration)	20	39
Septic ATN	9	17
SSG+PM induced AKI*	11	21
L-AMB induced AKI*	1	2
Glomerulonephritis	1	2
Not known	10	19
Stage of AKI at diagnosis (n = 52)
Stage 1	34	66
Stage 2	10	19
Stage 3	8	15
Active urinary sediments
Proteinuria (n = 38)
Yes	22	58
No	16	42
Hematuria (n = 38)
Yes	16	42
No	22	58
Pyuria (n = 37)
Yes	19	51
No	18	49
Outcome of AKI (n = 52)
Resolved	40	77
Not Resolved	5	9.6
Died	2	3.8
Unknown	5	9.6

AKI: Acute Kidney Injury, ATN: Acute Tubular Necrosis, L-AMB: liposomal amphotericin B.

*Drug induced AKI occurs post-admission.

Table 4

Rate of post-admission AKI per initial treatment allocation among patients with VL with baseline normal kidney function at LRTC between January 2019 –December 2019, Gondar, Ethiopia, N = 268.

Type of treatment	Post-admission AKI (n = 265*)		OR	p value
	No	Yes
SSG-baseda	206 (93%)	15 (7%)	1	0.13
L-AMB basedb	38 (86%)	6 (14%)	2.17 (0.8–6.0)

AKI: acute kidney injury, SSG: Sodium stibogluconate, PM: Paromomycin, L-AMB: liposomal amphotericin B; OR: odds ratio.

*Data missing for 3 patients.

aSSG-based: SSG+ PM (n = 219), SSG alone (n = 2).

bL-AMB-based: L-AMB alone (n = 40), L-AMB + Miltefosine (n = 4).