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RESEARCH ARTICLE
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Citation: Jacobo-Vargas TB, Báez-Saldaña R, Cruz-
Hervert LP, Fortoul TI, Ahumada-Topete VH,
Rodrı́guez-Ganén O, et al. (2023) Trimethoprim-
Sulfamethoxazole-associated early neutropenia in
Mexican adults living with HIV: A cohort study.
PLoS ONE 18(5): e0285541. https://doi.org/
10.1371/journal.pone.0285541
Editor: Dickens Otieno Onyango, Kisumu County,
KENYA
Received: December 18, 2022
Accepted: April 25, 2023
Published: May 11, 2023
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https://doi.org/10.1371/journal.pone.0285541
Copyright: © 2023 Jacobo-Vargas et al. This is an
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author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting information
files.
PLOS ONE | https://doi.org/10.1371/journal.pone.0285541 May 11, 2023
Trimethoprim-Sulfamethoxazole-associated
early neutropenia in Mexican adults living
with HIV: A cohort study
Thalia Berenice Jacobo-Vargas1,2, Renata Báez-Saldaña ID3*, Luis Pablo Cruz-Hervert4,5,
Teresa Imelda Fortoul6, Victor Hugo Ahumada-Topete ID7, Odalis Rodrı́guez-Ganén8,
Ricardo Stanley Vega-Barrientos3
1 Pharmacist in Pharmacology Research Unit and Hospital Pharmacy Department, National Institute of
Respiratory Diseases, Mexico City, Mexico, 2 Programa de Maestrı́a y Doctorado en Ciencias Médicas,
Odontológicas y de la Salud, National Autonomous University of Mexico, Mexico City, Mexico,
3 Hospitalization, National Institute of Respiratory Diseases, Mexico City, Mexico, 4 Professor of Orthodontic
Department in the Division of Postgraduate Studies and Research, Faculty of Dentistry, National Autonomous
University of Mexico, Mexico City, Mexico, 5 Program on Epidemiological and Emerging Risks, National
Autonomous University of Mexico, Mexico City, Mexico, 6 School of Medicine, National Autonomous
University of Mexico, Mexico City, Mexico, 7 Head of the Unit of Hospital Epidemiology and Infectious
Diseases, National Institute of Respiratory Diseases, Mexico City, Mexico, 8 Head of the Hospital Pharmacy
Department, National Institute of Respiratory Diseases, Mexico City, Mexico
* baezrd@unam.mx
Abstract
Introduction
Trimethoprim/sulfamethoxazole (TMP/SMX) is the antimicrobial of first choice in the treat-
ment and prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in immunocompromised
patients, particularly in people living with human immunodeficiency virus (HIV). TMP/SMX
use entails different adverse effects, and its association with early neutropenia is minimally
documented. This study aimed to identify the risk of early neutropenia associated with TMP/
SMX use in adults living with HIV in Mexico.
Methods
A prospective cohort study was conducted in TMP/SMX-naïve adults living with HIV admit-
ted to a third-level hospital between August 2019 and March 2020. Socio-demographic, clin-
ical, and laboratory data were collected. According to patients’ diagnostic, if they required
treatment or prophylaxis against PCP, medical staff decided to prescribe TMP/SMX, as it is
the first-line treatment. The risk of TMP/SMX induced early neutropenia, as well as associ-
ated factors were analyzed through a bivariate model and a multivariate Poisson regression
model. The strength of association was measured by incidence rate ratio (IRR) with 95%
confidence interval.
Results
57 patients were enrolled in the study, of whom 40 patients were in the TMP/SMX treat-
ment-group for treatment or prophylaxis of PCP (204.8 person-years of observation, median
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PLOS ONE
Funding: This research was supported by the
National Council of Science and Technology
(CONACyT). TBJ-V is a doctoral student from
Programa de Doctorado en Ciencias Médicas,
Odontológicas y de la Salud, National Autonomous
University of Mexico (UNAM), and received a
fellowship from CONACyT (CVU 1196087).
Competing interests: The authors have declared
that no competing interests exist.
PLOS ONE | https://doi.org/10.1371/journal.pone.0285541 May 11, 2023
Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study
26.5 days) and 17 patients were in the non-treatment group because they did not need the
drug for treatment or prophylaxis of PCP (87.0 person-years of observation, median 21
days). The incidence rate of early neutropenia in the TMP/SMX-treatment group versus
non-treatment group was 7.81 and 1.15 cases per 100 person-years, respectively. After
adjusting for stage 3 of HIV infection and neutrophil count <1,500 cells/mm3 at hospital
admission, the current use of TMP/SMX was not associated with an increase in the inci-
dence rate ratio of early neutropenia (adjusted IRR: 3.46; 95% CI: 0.25–47.55; p = 0.352).
Conclusions
The current use of TMP/SMX in Mexican adults living with HIV was not associated with an
increase in the incidence rate ratio of early neutropenia.
Introduction
Trimethoprim/sulfamethoxazole (TMP/SMX) is the first antimicrobial of choice in the treat-
ment and prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in immunocompromised
patients, especially in those living with Human Immunodeficiency Virus (HIV). Although the
introduction of an antiretroviral therapy (ART) has reduced the morbidity and mortality asso-
ciated with opportunistic infections, PCP remains one of the most common diagnoses in
patients living with HIV [1].
TMP/SMX synergistically blocks bacterial and fungal synthesis of folic acid, a vital cofactor
in the production of thymidine and purines [2, 3]. In terms of safety, it is known that people
living with HIV have a high frequency of adverse reactions due to TMP/SMX use (20–85%). In
addition to different types of cytopenia (anemia, neutropenia, and thrombocytopenia), the
appearance of which can range from 6 days to 5 weeks (36), rash (30–55%), elevation of liver
enzymes (20%) and of creatinine (1–5%), hyperkalemia, and hyponatremia may occur, usually
around day 7 to 14 of treatment [4, 5].
Studies reporting the association of neutropenia with TMP/SMX use are limited. In a cross-
sectional study, Fekene et al. (2018) reported a significant association between prophylactic
use of TMP/SMX and leukopenia (Odds Ratio (OR) 2.34, 95% CI 1.05–5.19, p = 0.036) [6]; on
the other hand, Munyazesa et al. (2012) did not find a significant association of TMP/SMX
with moderate neutropenia (OR 5.69, 95% CI 0.63–51.45; p = 0.122) [7]. While Moh et al.
(2005) found that grade 3–4 neutropenia was higher in patients with positive serum hepatitis B
antigen at baseline (HR 1.58, 95% CI 1.00–2.52; p = 0.05), and compared with patients with a
baseline neutrophil count �1500 cells/mm3, those with baseline neutrophils al 750–999 cells/
mm3 and 1000–1499 cells/mm3 had an HR of grade 3–4 of neutropenia of 3.24 (95% CI 1.94–
5.42; p< 0.001) and 2.31 (95% CI 1.55–3.44; p< 0.001), respectively [8]. And in a 6-year longi-
tudinal follow-up study of a cohort of patients living with HIV receiving TMP/SMX prophy-
laxis, the authors found that the only variable associated with neutropenia was a low baseline
CD4 T-cell count [9], which determine the stage of HIV infection. In Mexico, we only found
one study in 15 patients living with HIV which found that TMP/SMX dose over 160/800 milli-
grams respectively, was a risk factor for dermatological adverse reactions to TMP/SMX (OR
12.7, 95% CI 1.59–102.7; p = 0.017) but does not mention whether the patients were new
TMP/SMX users [10].
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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

No studies have been conducted in Mexican population, even though neutropenia may be a
risk factor for serious bacterial infections. Thus, the objective of this study was to identify the
risk of early neutropenia caused by TMP/SMX use in adult patients living with HIV in a third-
level hospital in Mexico City.

Methods
Study design
A prospective cohort design was used to assess the differences in the risk of early neutropenia
between TMP/SMX-treatment group and non-treatment group for prophylaxis or treatment
of PCP during the period of current use (while taking the drug), in adults living with HIV hos-
pitalized between August 2019 and March 2020 at the National Institute of Respiratory Dis-
eases “Ismael Cosio Villegas” (INER) in Mexico City. We selected hospitalized patients as our
study population because complete blood count (CBC) studies are performed almost daily in
the hospital, and we need this information to identify neutropenia events occurring early after
initiation of TMP/SMX. To control for the possible bias of being hospitalized, in the multivari-
ate analysis we considered the variable stage 3 of HIV infection at hospital admission as a
potential confounder.

Cohort definition and data collection

Inclusion criteria were hospitalized patient aged 18 years or older, with confirmed diagnosis of
HIV, without neutropenia at the start of the study, or with grade 1 or grade 2 neutropenia
according to the Common Terminology Criteria for Adverse Events classification version 5.0
[11] and with no TMP/SMX prescription in the 6 months prior to start date (all patients were
TMP/SMX-naïve or new users). This washout period was selected to ensure that patients pre-
viously exposed to TMP/SMX returned to a naïve state. Patients who had used any chemother-
apeutic drug during the past 6 months before the beginning of the study were excluded, as well
as those with chronic kidney disease or acute-on-chronic kidney disease, severe aplastic ane-
mia or agranulocytosis and with less than 3 days of TMP/SMX use. Patients who did not have
CBC studies done at least 10 days after the start of follow-up were also excluded, because cyto-
penias are reported to occur in 6 days to 5 weeks [12], and we focused on studying early neu-
tropenia events occurring soon after the start of TMP/SMX, within the first 5 weeks, for
treatment or prophylaxis of PCP.
Previous exposure to TMP/SMX or any chemotherapeutic drug during the past 6 months
before the beginning of the study was identified by patient interview and by reviewing clinical
records because there are not electronic healthcare databases in different health services nor
between them.
According to patients’ diagnostic, if they required treatment or prophylaxis against PCP,
medical staff decided to prescribe TMP/SMX, as it is the first-line treatment. The definition of
the TMP/SMX-treatment group were patients with TMP/SMX administered at any dose for at
least 3 days, intravenous and/or oral, according to the manual administration records as the
source of information at the hospital. Non-treatment group were patients without any TMP/
SMX dose administration because they did not have a diagnosis of PCP and therefore did no
need TMP/SMX. Patients in the TMP/SMX-treatment group were excluded from entering the
non-treatment group at any time.
Consecutive sampling was used to select patients who met the inclusion criteria. Patient
information was obtained from daily manual review of prescription and administration rec-
ords and during the medical visit from Monday to Friday to record drug use, results of labora-
tory studies, diagnosis, and clinical evolution. Follow up after hospital discharge was done

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

through monthly telephone calls to identify drug use, and in the case of TMP/SMX-treatment
group, the date of drug suspension.
The definition of neutropenia case for patients without baseline neutropenia was neutrophil
count <1,500/mm3, and for patients with neutropenia at baseline, either grade 1 (neutrophil
count �1,500/mm3) or grade 2 (neutrophil count <1,500 to 1,000/mm3) was a 20% decrease
with respect to the initial neutrophil count. The index date of neutropenia cases was the first
date on which neutrophils were found to have a value that met the case definition. CBC was
done at the INER laboratory in Beckman Coulter’s UniCel1 DxH 800 Coulter1 Cellular Anal-
ysis System with an automated, validated method using the impedance technique. The grades
of neutropenia for the identified cases were defined according to the absolute neutrophil count
using the Common Terminology Criteria for Adverse Events classification, version 5.0 as fol-
lows: grade 2, neutrophils <1,500 to 1,000/mm3; grade 3, neutrophils <1,000 to 500/mm3; and
grade 4, neutrophils <500/mm3 [11].
Information bias (detection) was ruled out because the comparison between the groups
with and without early neutropenia showed no statistically significant difference between the
number of days of CBC testing (6 days [4–9 days] vs. 5 days [3–7.5 days], p = 0.0981).
Glomerular filtration rate (GFR) was recorded as a quantitative variable; then a categorical
variable was generated depending on whether or not GFR met the condition of reaching <30
ml / min at patient’s admission to the hospital, because from this rate, dose adjustment is
required for some drugs that are eliminated via the kidney, such as TMP/SMX. CD4 T-cell
count was recorded as a quantitative variable throughout the follow-up, and a dichotomous
variable was generated if the lymphocyte count at hospital admission was <200 cells/mm3,
since this condition determines the definition of stage 3 of VIH infection, which entails an
increased risk of more serious opportunistic infections [13]. Diagnoses as well as prescribed
and administered medications were identified throughout hospitalization and until hospital
discharge. Drugs given as single doses were not considered because when administered once,
it is unlikely that they are involved in blood abnormalities. S1 Appendix describes the rest of
the variables that were included in the initial exploratory analysis.

Statistical analysis
The descriptive analysis was carried out depending on the type of variable and its distribution,
evaluated by the Shapiro-Wilk test. The continuous variables were presented as
mean ± standard deviation (SD) or median (IQR), as appropriate. The incidence rate of early
neutropenia was calculated in number of cases per 100 person-years of follow-up.
According to the distribution of the variables involved, the continuous variables (age, body
mass index (BMI), hospital stay, GFR, viral load, CD4 T-cell count, number of days of CBC
testing) were compared with the dependent variable (neutropenia) using the t-student test or
Mann-Whitney U test, whereas the categorical variables (sex, acute kidney injury, neutrophil
count <1,500 cells/mm3 at admission, risk group for HIV infection, stage 3 of HIV at admis-
sion, sepsis, ART at admission, ganciclovir/valganciclovir use) were compared with the depen-
dent variable (neutropenia) using the X2 test or Fisher’s exact test.
The association between TMP/SMX use and early neutropenia was estimated using robust
Poisson regression model expressed as IRR with 95% confidence interval, adjusted for poten-
tial confounder such as stage 3 of HIV infection at hospital admission [4, 14], and for neutro-
phil count <1,500 cells/mm3 at hospital admission because basal neutropenia has been
identified as a risk factor for the development of a more severe degree of neutropenia [8].
Data were analyzed using STATA statistical package version 14 (StataCorp LP, College Sta-
tion, TX, USA).

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

Sample size calculation

With 40 patients in the TMP/SMX-treatment group, 17 patients in the non-treatment group,
neutropenia in TMP/SMX-treatment group of 40% and in non-treatment group of 5.9%, the
power calculation for the cohort study with OpenEpi was 74% [15].

Ethical considerations
The study was approved by the Ethics in Researchg Committee and the Research Committee
of the Ismael Cosio Villegas National Institute of Respiratory Diseases (registration number
E10-19). An informed consent form was signed by each study participant.

Results
Socio-demographic and clinical characteristics of the study cohort
57 patients were enrolled in the study, of whom 40 patients were in the TMP/SMX-treatment
group for treatment or prophylaxis of PCP and 17 patients were in the non-treatment group
because they did not need the drug for treatment or prophylaxis of PCP (Fig 1). Mean age was
38.37 ± 10.28 years. Most participants were men (92.98%), BMI was within the normal cate-
gory reference range (22.62 ± 3.84). Median (IQR) time of HIV diagnosis was 3.7 months (0–
82 months), 70.18% were at the HIV risk group of men who have sex with men (MSM),
75.44% of patients were admitted to the hospital with stage 3 of HIV infection. 17.54% of
patients (n = 10) presented sepsis during hospitalization and only one patient had positive
serum hepatitis B antigen at baseline. There was statistically significant difference between
TMP/SMX-treatment group and non-treatment group in variables such as admission in stage
3 of HIV infection, viral load and CD4 T-cell count at admission, number of days of CBC test-
ing during follow-up, and ART at hospital admission (Table 1).

Socio-demographic and clinical characteristics between the groups with

and without early neutropenia
Of the 57 patients of the study cohort, 17 patients developed early neutropenia during the fol-
low-up. The median (IQR) time of HIV diagnosis was 0.17 months (0–74.3 months) in the
early neutropenia group and 4.78 months (0–84.07 months) in the non-neutropenia group
(p = 0.523). 94.12% of individuals in the early neutropenia group were admitted with stage 3 of
HIV infection vs 67.5% in the non-neutropenia group (p = 0.044). Of the 57 patients, only
24.6% (n = 14), belonging to the group without early neutropenia, were on ART at admission.
29.41% of patients in the early neutropenia group presented sepsis during hospitalization vs
12.5% in the non-neutropenia group (p = 0.145) (Table 2).

Measures of frequency and association of early neutropenia

The 57 patients of the study cohort were followed for 291.84 person-years (median of 22 days,
maximum of 111 days). 17 patients developed early neutropenia (29.82%); of them, in the
non-treatment group (87.04 person-years of follow-up, median of 29 days and maximum of
103 days), one case of grade 3 neutropenia was identified. The TMP/SMX-treatment group
(204.80 person-years of follow-up, median of 12 days and maximum of 111 days of follow-up)
comprised the remaining 16 cases of early neutropenia, of which 12 cases were grade 2, 3 cases
were grade 3, and 1 case was grade 4 neutropenia. The incidence rate of early neutropenia in
the TMP/SMX-treatment group and non-treatment group was 7.81 and 1.15 cases per 100 per-
son-years, respectively (Table 3).

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

Fig 1. Flow diagram of patient selection and follow-up.

https://doi.org/10.1371/journal.pone.0285541.g001

After adjusting for stage 3 of HIV infection and neutrophil count <1,500 cells/mm3 at hos-
pital admission, current use of TMP/SMX was not associated with an increase in the incidence
rate ratio of early neutropenia (adjusted IRR: 3.46; 95% CI: 0.25–47.55; p = 0.352) (Table 4).
Other clinically relevant variables, such as sepsis during hospitalization as potential con-
founder, and treatment with ganciclovir/valganciclovir during hospitalization as a drug
involved in cytopenias, were not include in the multivariate model because they did not result
in a statistically significant difference between the groups with and without early neutropenia
in the bivariate model (Table 2). Nor was the variable CD4 T-cell count <200 cells/mm3 at
admission included in the multivariate model, because 100% of the patients with early neutro-
penia (17 cases) had this condition at hospital admission and we have already considered a
related variable, stage C3 of HIV infection at hospital admission.

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

Table 1. Socio-demographic and clinical characteristics of the total cohort and between TMP/SMX-treatment group and non-treatment group.
Total N = 57 TMP/SMX-treatment group (n = 40) Non-treatment group (n = 17) P value
Age [years], Mean (SD) 38.37 (10.28) 37.25 (9.22) 41 (12.32) 0.2701
Men sex, n (%) 53 (92.98) 37 (92.50) 16 (94.12) 1.000
BMI [kg/m2], Mean (SD) 22.62 (3.84) 21.98 (3.69) 24.11 (3.88) 0.0539
Hospital stay [days], Median (IQR) 13 (10,15) 12 (9.50,16.50) 14 (11,15) 0.7725
GFR at admission [ml/min], Median (IQR) 102 (86,121.44) 105 (83.45,122.93) 95.35 (88.45,104.20) 0.2221
Acute kidney injury at admission, n (%) 4 (7.02) 3 (7.50) 1 (5.88) 1.000
Neutrophil count <1,500 cells/mm3 at admission, n (%) 7 (12.28) 7 (17.50) 0 0.091
Risk group MSM, n (%) 40 (70.18) 30 (75) 10 (58.82) 0.222
Stage 3 of HIV at admission, n (%) 43 (75.44) 38 (95) 5 (29.41) 0.000
Viral load at admission [copies/ml], Median (IQR) 79055 (3869,403558) 212754.50 (52177,479871) 47 (1,4117) 0.0000
CD4 T-cell count at admission [cells/mm3], Median (IQR) 61 (20,188) 37.50 (19,89.50) 301 (188,636) 0.0000
3
CD4 T-cell <200 cells/ mm at admission, n (%) 44 (77.19) 38 (95.0) 6 (35.29) 0.000
Sepsis, n (%) 10 (17.54) 9 (22.50) 1 (5.88) 0.253
Documented bacterial infection, n (%) 25 (43.86) 16 (40.0) 9 (52.94) 0.368
Parasitic infection, n (%) 4 (7.02) 4 (10.0) 0 0.306
Number of days of CBC testing, Median (IQR) 5 (3,8) 5.50 (4,9) 3 (3,5) 0.0009
ART at admission, n (%) 14 (24.56) 1 (2.50) 13 (76.47) 0.000
Ganciclovir/Valganciclovir use, n (%) 6 (10.5) 5 (12.5) 1 (5.88) 0.657

Abbreviations: ART: Antiretroviral therapy; BMI: Body mass index; CBC: Complete blood count; IQR: Inter quartile range; MSM: men who have sex with men; SD:
standard deviation; TMP/SMX: trimethoprim/ sulfamethoxazole.
P values were obtained from X2 or Fisher’s exact test for categorical variables and t-Student or Mann-Whitney U test for continuous variables.

https://doi.org/10.1371/journal.pone.0285541.t001

Table 2. Socio-demographic and clinical characteristics of the total cohort and between the groups with and without early neutropenia.
Total N = 57 With neutropenia (n = 17) Without neutropenia (n = 40) P value
Age [years], Mean (SD) 38.37 (10.28) 36.35 (10.36) 39.23 (10.25) 0.3389
Men sex, n (%) 53 (92.98) 16 (94.12) 37 (92.50) 1.000
BMI [kg/m2], Mean (SD) 22.62 (3.84) 21.16 (3.62) 23.23 (3.81) 0.0620
Hospital stay [days], Median (IQR) 13 (10,15) 12 (9,15) 13 (11,16) 0.6934
GFR at admission [ml/min], Median (IQR) 102 (86,121.44) 116.0 (81,122.26) 97.5 (86.75,116.4) 0.4073
Acute kidney injury at admission, n (%) 4 (7.02) 2 (11.76) 2 (5.0) 0.575
Neutrophil count <1,500 cells/mm3 at admission, n (%) 7 (12.28) 4 (23.53) 3 (7.50) 0.180
Risk group MSM, n (%) 40 (70.18) 11 (64.71) 29 (72.50) 0.547
Stage 3 of HIV infection at admission, n (%) 43 (75.44) 16 (94.12) 27 (67.50) 0.044
Viral load at admission [copies/ml], Median (IQR) 79055 (3869,403558) 197726 (51256,315974) 50613 (46,465986.5) 0.2387
CD4 T-cell count at admission [cells/mm3], Median (IQR) 61 (20,188) 45 (18,84) 107.5 (23,284.50) 0.0292
CD4 T-cell <200 cells/mm3 at admission, n (%) 44 (77.19) 17 (100.0) 27 (67.50) 0.006
Sepsis, n (%) 10 (17.54) 5 (29.41) 5 (12.50) 0.145
Documented bacterial infection, n (%) 25 (43.86) 5 (29.41) 20 (50.0) 0.152
Parasitic infection, n (%) 4 (7.02) 2 (11.76) 2 (5.0) 0.575
Number of days of CBC testing, Median (IQR) 5 (3,8) 6 (4,9) 5 (3,7.50) 0.0981
ART at admission, n (%) 14 (24.56) 0 14 (35.0) 0.005
Ganciclovir/Valganciclovir use, n (%) 6 (10.5) 2 (11.76) 4 (10.0) 1.000

Abbreviations: ART: Antiretroviral therapy; BMI: Body mass index; CBC: Complete blood count; MSM: men who have sex with men.
P values were obtained from X2 or Fisher’s exact test for categorical variables and t-Student or Mann-Whitney U test for continuous variables.

https://doi.org/10.1371/journal.pone.0285541.t002

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

Table 3. Incidence rate and incidence rate ratio of early neutropenia.

Neutropenia cases Person-time Incidence rate (per 100 person- Crude IRR (95% CI)
years)
Neutropenia 291.84 person- TMP/SMX-treatment 16 204.80 person- 7.81 6.79 (0.96–48.09)
years group years
Non-treatment group 1 87.04 person-years 1.15 Ref

Abbreviations: 95% CI: 95% Confidence Interval; IRR: Incidence rate ratio; TMP/SMX: trimethoprim/ sulfamethoxazole.

https://doi.org/10.1371/journal.pone.0285541.t003

A post hoc power analysis was conducted using an online calculator for “A-priori Sample
Size for Multiple Regression” [16] to know the minimum required sample size. Considering
an anticipated effect size of 0.1, 80% of desired statistical power level, 3 predictors and α =
0.05, the minimum required sample size resulted in 112.

Discussion
This is the first study to report an estimate of the incidence rate and incidence rate ratio of
TMP/SMX-associated early neutropenia in Mexican adults living with HIV. We identified an
incidence rate of 7.81 cases of early neutropenia per 100 person-years in the TMP/SMX-treat-
ment group. After adjusting for stage 3 of HIV infection and neutrophil count <1,500 cells/
mm3 at hospital admission we found that the current use of TMP/SMX is not associated with
an increase in the incidence rate ratio of early neutropenia (adjusted IRR: 3.46; 95% CI: 0.25–
47.55; p = 0.352).
TMP/SMX has been reported to cause neutropenia due to folate deficiency [17]; however,
the mechanism has not yet been fully clarified [18]. We found a frequency of neutropenia of
29.82%, similar to that identified by Tamir et al. in 2019, who reported a frequency of leukope-
nia of 24.4% [14]. The difference may be due to variations in the methodology and definitions
of cytopenia, as well as different clinical and sociodemographic characteristics of the studied
population. In addition, these authors indicate that patients with low CD4 T-cell count, high
viral loads, and advanced stages of HIV infection present an increase in the prevalence of leu-
kopenia, which is consistent with our findings, since 100% of patients who developed early
neutropenia had CD4 T-cell < 200 cells / mm3 at hospital admission, the median viral load at
admission was almost 4 times higher in the group that developed early neutropenia vs the
group that did not develop it, and 94% of patients in the early neutropenia group were admit-
ted in stage 3 of HIV infection, unlike the group without early neutropenia, where only 67.5%
were admitted in that stage of HIV infection [14]. This indicates that the group of patients who
developed early neutropenia had greater degree of immunosuppression at hospital admission.
Although the current use of TMP/SMX in Mexican patients living with HIV was not associ-
ated with an increased incidence rate ratio of early neutropenia (adjusted IRR of early neutro-
penia 3.46; 95% CI 0.25–47.55; p = 0.352), they agree in the direction of the association with

Table 4. Unadjusted and adjusted measures of association of early neutropenia.

Unadjusted IRRa (95% CI) P value Adjusted IRRa (95% CI) P value
TMP/SMX 6.80 (0.96–48.09) 0.055 3.46 (0.25–47.55) 0.352
Neutrophil count <1,500 cells/mm3 at admission 2.20 (0.99–4.90) 0.054 1.91 (0.87–4.20) 0.107
Stage 3 of HIV infection at admission 5.21 (0.74–36.44) 0.096 2.65 (0.26–26.76) 0.409

Abbreviations: 95% CI: 95% Confidence Interval; IRR: Incidence rate ratio; TMP/SMX: trimethoprim/ sulfamethoxazole.
a
Robust Poisson regression.

https://doi.org/10.1371/journal.pone.0285541.t004

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

those reported by Fekene et al. [6], who found in a quantitative cross-sectional study that the
prophylactic use of TMP/SMX in adults living with HIV was associated with increased risk of
leukopenia (OR 2.34, 95% CI 1.05–5.19, p = 0.036), considering that neutrophils constitute
40–70% of the total leucocyte count [6]. Our results also agree with Munyazesa et al., who did
not found association of TMP/SMX and/or dapsone with moderate neutropenia (OR 5.69,
95% CI 0.63–51.45; p = 0.122) [7].
The possible confusion by indication should be considered when finding such associations
between antibacterial agents and blood dyscrasias, because antibacterial drugs can be pre-
scribed for the treatment of an infection among whose clinical manifestations are blood dys-
crasias [6]. Yet, in the present study, confusion by indication can be ruled out, since PCP
pneumonia, for which TMP/SMX was indicated, does not have neutropenia as a clinical
manifestation.
Due to the small sample size, we only selected two variables to adjust the IRR of TMP/SMX
associated early neutropenia in the multivariate model. Neutrophil count <1,500 cells / mm3
at hospital admission was considered because in the bivariate analysis between the groups with
and without early neutropenia, this variable obtained a p = 0.180, also, it modified in >10%
the IRR of early neutropenia due to TMP/SMX use (3.02 vs. 4.75), and baseline neutrophil
count <1,500 cells / mm3 has been reported to increase the risk of more severe neutropenia
(HR 2.31, 95% CI 1.55–3.44; p<0.001) [8]. Additionally, stage 3 of HIV infection at admission
was included in the multivariate model, since the advanced stages of HIV infection have been
found to be significantly associated with an increase in the prevalence of anemia, leukopenia,
and thrombocytopenia [14], and in the bivariate analysis between the groups with and without
early neutropenia, stage 3 of HIV infection at admission obtained a p = 0.044. Instead, CD4 T-
cell count <200 cells/mm3 was not considered in the adjusted early neutropenia risk model,
despite being a confounding factor because 100% of the early neutropenia cases had CD4 T-
cell count <200 cells/mm3 at hospital admission, which is consistent with a 6-year longitudinal
follow-up study of a cohort of patients living with HIV that used prophylactic TMP/SMX
where authors found that the only variable associated with neutropenia was a low baseline
CD4 T-cell count [9].
Among the limitations of this study, data about previous medication use could be left trun-
cated, resulting in the incomplete capture of medical history and previous use of medications
because there are not healthcare databases in different health services nor between them; this
truncation is partially mitigated by comparing information from clinical records with patient
interviews. Also, as follow-up for TMP/SMX-treatment group and non-treatment group was
carried out until the first neutropenia event occurred or to the time of the last laboratory stud-
ies that included CBC, there is potential right truncation because we do not have information
of CBC after the discharge of the patients because of the hospital reconversion due to the
COVID-19 pandemic. However, the time of follow-up allowed us to identify early neutropenia
events. Another limitation of the study is that most patients had a CD4 T-cell count <200
cells/mm3 at hospital admission (77.19%); they came with a high degree of immunosuppres-
sion and little control of HIV infection, so the results cannot be generalized to other patients
who do not meet these characteristics. Other limitation of the study is the low power obtained
for the cohort study (74%) due to the small size of the cohort, as patient enrollment could not
continue due to the COVID-19 hospital conversion, suggesting that our true power is even
lower for multivariate analysis. However, based on the R2 value of the multivariate model
(0.0997), we calculated a small effect size of 0.11 [19]. Although our effect size is small, we can
infer that the risk of not identifying a possible effect is very low and additionally, another study
has reported no significant effect as mentioned previously. These findings emphasize the need
for more studies to identify whether there is a clinically relevance. Furthermore, follow-up

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

could not be done for a longer time to also identify the risk of neutropenia in the exposure
periods of "recent use" and "past use" in addition to "current use". Lastly, the study was devel-
oped in a single reference center (unicentric). The early neutropenia association with current
TMP/SMX use found in this study may thus be overestimated because of the previous
limitations.
Regarding applicability of our estimates to other adult patients living with HIV, even
though INER patients included in our cohort study, constitute a different population than that
found in other health institutions as INER is a national reference hospital and most patients
are admitted with a significant deterioration in their health status, other less advanced stages
of HIV infection were also included in addition to stage 3, as well as very varied baseline viral
loads, including undetectable patients, both men and women, with a wide age range and from
different states of Mexico. All this means that the model can be applied to patients with the
variety of characteristics above mentioned.
The results of this study highlight the need to identify groups at high-risk of developing
early neutropenia among patients living with HIV with TMP/SMX-treatment. This will allow
early pharmacological adjustments and timely initiation of granulocyte colony-stimulating fac-
tor therapies to prevent neutropenia or its worsening, which can have fatal consequences. Fur-
thermore, the study contributes to providing evidence that is scarce in the literature on
measures of frequency and association of early neutropenia due to TMP/SMX use, even
though neutropenia was reported as the most frequent short-term adverse event in a trial of
prophylactic TMP/SMX in African adults living with HIV [20]. Finally, our study contributes
to the development of pharmacoepidemiology in Mexico.

Conclusion
The current use of TMP/SMX in Mexican patients living with HIV was not associated with an
increased in the incidence rate ratio of early neutropenia. This study highlights important gaps
in measures of association of TMP/SMX-associated neutropenia in adults living with HIV. We
encourage researchers to continue investigating this area to determine if there is a significant
effect that could result in clinical benefit.

Supporting information
S1 Appendix. Collected independent variables.
(DOCX)
S2 Appendix. STROBE checklist cohort. STROBE Statement—Checklist of items that should
be included in reports of cohort studies [1].
(DOCX)
S3 Appendix. Study database.
(DTA)

Acknowledgments
We would like to thank Dr Gustavo Reyes Teran, Head of the Coordinating Commission of
National Institutes of Health and High Specialty Hospitals in Mexico City, Mexico, and to Dr
Francisco Javier Flores Murrieta and Dra Miriam del Carmen Carrasco Portugal from
National Institute of Respiratory Diseases, Mexico City, Mexico, for all the support given to
carry out this study.

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PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

Thalia Berenice Jacobo-Vargas is student from the program Doctorado en Ciencias Médi-
cas, Odontológicas y de la Salud at the National Autonomous University of Mexico, Mexico
City, Mexico, and is the recipient of scholarship from CONACyT, Mexico (CVU 1196087).

Author Contributions
Conceptualization: Thalia Berenice Jacobo-Vargas, Renata Báez-Saldaña, Luis Pablo
Cruz-Hervert, Teresa Imelda Fortoul.
Data curation: Thalia Berenice Jacobo-Vargas, Luis Pablo Cruz-Hervert.
Formal analysis: Thalia Berenice Jacobo-Vargas, Renata Báez-Saldaña, Luis Pablo
Cruz-Hervert, Teresa Imelda Fortoul.
Investigation: Thalia Berenice Jacobo-Vargas, Renata Báez-Saldaña, Luis Pablo Cruz-Hervert,
Teresa Imelda Fortoul, Victor Hugo Ahumada-Topete.
Methodology: Thalia Berenice Jacobo-Vargas, Renata Báez-Saldaña, Luis Pablo Cruz-Hervert,
Teresa Imelda Fortoul.
Project administration: Thalia Berenice Jacobo-Vargas, Renata Báez-Saldaña, Luis Pablo
Cruz-Hervert, Teresa Imelda Fortoul.
Resources: Thalia Berenice Jacobo-Vargas, Victor Hugo Ahumada-Topete, Odalis
Rodrı́guez-Ganén, Ricardo Stanley Vega-Barrientos.
Supervision: Renata Báez-Saldaña, Luis Pablo Cruz-Hervert, Teresa Imelda Fortoul, Victor
Hugo Ahumada-Topete, Odalis Rodrı́guez-Ganén, Ricardo Stanley Vega-Barrientos.
Visualization: Luis Pablo Cruz-Hervert.
Writing – original draft: Thalia Berenice Jacobo-Vargas, Renata Báez-Saldaña, Luis Pablo
Cruz-Hervert, Teresa Imelda Fortoul, Victor Hugo Ahumada-Topete.
Writing – review & editing: Thalia Berenice Jacobo-Vargas, Renata Báez-Saldaña, Luis Pablo
Cruz-Hervert, Teresa Imelda Fortoul, Victor Hugo Ahumada-Topete, Odalis
Rodrı́guez-Ganén, Ricardo Stanley Vega-Barrientos.

References
1. Siegel M, Masur H, Kovacs J. Pneumocystis jirovecii Pneumonia in Human Immunodeficiency Virus
Infection. Semin Respir Crit Care Med. 2016; 37(2):243–56. https://doi.org/10.1055/s-0036-1579556
PMID: 26974301
2. Currier JS, Havlir DV. CROI 2017: Complications and Comorbidities of HIV Disease and Its Treatment.
Top Antivir Med. 2017 May/Jun; 25(2):77–83. PMID: 28598792
3. Church JA, Fitzgerald F, Walker AS, Gibb DM, Prendergast AJ. The expanding role of co-trimoxazole in
developing countries. Lancet Infect Dis. 2015; 15(3):327–39. https://doi.org/10.1016/S1473-3099(14)
71011-4 Epub 2015 Jan 21. Erratum in: Lancet Infect Dis. 2015; 15(3):263. PMID: 25618179
4. Kaplan JE, Benson C, Holmes KH, Brooks JT, Pau A, Masur H. Guidelines for prevention and treatment
of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the
National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of
America. MMWR Recomm Rep. 2009; 58:1–207. quiz CE1–4.
5. Spanish Agency of Medicines and Medical Devices (AEMPS). Technical Information Soltrim (trimetho-
prim sulfamethoxazole) 160/800 mg injectable solution. [Cited 24 August 2022]. In: AEMPS [Internet].
https://cima.aemps.es/cima/dochtml/ft/54920/FichaTecnica_54920.html
6. Fekene TE, Juhar LH, Mengesha CH, Worku DK. Prevalence of cytopenias in both HAART and
HAART naïve HIV infected adult patients in Ethiopia: a cross sectional study. BMC Hematol. 2018;
18:8. https://doi.org/10.1186/s12878-018-0102-7 PMID: 29632668

PLOS ONE | https://doi.org/10.1371/journal.pone.0285541 May 11, 2023 11 / 12

PLOS ONE Trimethoprim-Sulfamethoxazole-associated early neutropenia in Mexican adults living with HIV: A cohort study

7. Munyazesa E, Emile I, Mutimura E, Hoover DR, Shi Q, McGinn AP, et al. Assessment of haematologi-
cal parameters in HIV-infected and uninfected Rwandan women: a cross-sectional study. BMJ Open.
2012; 2:e001600. https://doi.org/10.1136/bmjopen-2012-001600 PMID: 23169875
8. Moh R, Danel C, Sorho S, Sauvageot D, Anzian A, Minga A, et al. Haematological changes in adults
receiving a zidovudine-containing HAART regimen in combination with cotrimoxazole in Côte d’Ivoire.
Antivir Ther. 2005; 10(5):615–24. https://doi.org/10.1177/135965350501000510 PMID: 16152755
9. Toure S, Gabillard D, Inwoley A, Seyler C, Gourvellec G, Anglaret X. Incidence of neutropenia in HIV-
infected African adults receiving co-trimoxazole prophylaxis: a 6-year cohort study in Abidjan, Côte
d’Ivoire. Trans R Soc Trop Med Hyg. 2006; 100(8): 785–90. https://doi.org/10.1016/j.trstmh.2005.11.
008 PMID: 16458337
10. Martı́nez-Martı́nez MC, Hernández-Morales MR, Mancilla-Hernández E. The frequency of adverse
reactions to sulfamethoxazole with trimethoprim and risk factors in VIH patients. Rev Alerg Mex. 2020;
67(2):96–101. https://doi.org/10.29262/ram.v67i2.670 PMID: 32892524
11. National Institutes of Health (NIH). National Cancer Institute. Common Terminology Criteria for Adverse
Events (CTAE) v5.0. [Cited 04 de June 2019]. In: NIH [Internet]. https://ctep.cancer.gov/
protocoldevelopment/electronic_applications/ctc.htm
12. Myers MW, Jick H. Hospitalization for serious blood and skin disorders following co-trimoxazole. Br J
Clin Pharmacol. 1997; 43(6):649–651. https://doi.org/10.1046/j.1365-2125.1997.00590.x PMID:
9205827
13. Deeks S, Overbaugh J, Phillips A, Buchbinder S. HIV infection. Nat Rev Dis Primers. 2015; 1;1:15035.
https://doi.org/10.1038/nrdp.2015.35 PMID: 27188527
14. Tamir Z, Seid A, Haileslassie H. Magnitude and associated factors of cytopenias among antiretroviral
therapy naïve Human Immunodeficiency Virus infected adults in Dessie, Northeast Ethiopia. PLoS
ONE. 2019; 14(2):e0211708. https://doi.org/10.1371/journal.pone.0211708 PMID: 30759131
15. Dean AG, Sullivan KM, Soe MM. (2013). OpenEpi: Open Source Epidemiologic Statistics for Public
Health [Software]. [Cited 4 March 2023]. www.OpenEpi.com
16. Soper, D.S. (2022). Free Statistics Calculators version 4.0. A-priori Sample Size Calculator for Multiple
Regression [Software]. [Cited 23 November 2022]. https://www.danielsoper.com/statcalc/calculator.
aspx?id=1
17. Ho JM, Juurlink DN. Considerations when prescribing trimethoprim-sulfamethoxazole. CMAJ. 2011;
183(16):1851–8. https://doi.org/10.1503/cmaj.111152 PMID: 21989472
18. Andrès E, Mourot-Cottet R. Non-chemotherapy drug-induced neutropenia—an update. Expert Opin
Drug Saf. 2017; 16(11):1235–1242. https://doi.org/10.1080/14740338.2017.1376645 PMID: 28879784
19. Soper, D.S. (2023). Free Statistics Calculators version 4.0. Effect Size Calculator for Multiple Regres-
sion [Software]. [Cited 20 March 2023]. https://www.danielsoper.com/statcalc/calc
20. Anglaret X, Chêne G, Attia A, Toure S, Lafont S, Combe P, et al. Early chemoprophylaxis with trimetho-
prim—sulphamethoxazole for HIV-1- infected adults in Abidjan, Côte d’Ivoire: a randomised trial.
Cotrimo-CI Study Group. Lancet. 1999; 353(9163):1463–8. https://doi.org/10.1016/s0140-6736(98)
07399-1 PMID: 10232311.

PLOS ONE | https://doi.org/10.1371/journal.pone.0285541 May 11, 2023 12 / 12