International Journal of

Environmental Research
and Public Health

Article
Causes of Moderate and Severe Anaemia in a High-HIV and
TB-Prevalent Adult Population in the Eastern Cape Province,
South Africa
Don O’Mahony 1, * , Sikhumbuzo A. Mabunda 2,3,4 , Mbulelo Mntonintshi 1 , Joshua Iruedo 1 ,
Ramprakash Kaswa 1 , Ernesto Blanco-Blanco 5 , Basil Ogunsanwo 6 , Kakia Anne Faith Namugenyi 6 ,
Sandeep Vasaikar 7 and Parimalaranie Yogeswaran 1

1 Department of Family Medicine and Rural Health, Walter Sisulu University, Mthatha 5117, South Africa
2 School of Population Health, University of New South Wales, Sydney 2052, Australia
3 George Institute for Global Health, University of New South Wales, Sydney 2042, Australia
4 Department of Public Health, Walter Sisulu University, Mthatha 5117, South Africa
5 Department of Laboratory Medicine and Pathology, Walter Sisulu University, Mthatha 5100, South Africa
6 Department of Surgery, Walter Sisulu University, Mthatha 5117, South Africa
7 Department of Microbiology, Walter Sisulu University, Mthatha 5117, South Africa
* Correspondence: donomahony@gmail.com

Citation: O’Mahony, D.; Mabunda,
S.A.; Mntonintshi, M.; Iruedo, J.;
Kaswa, R.; Blanco-Blanco, E.;
Ogunsanwo, B.; Namugenyi, K.A.F.;
Vasaikar, S.; Yogeswaran, P. Causes of
Moderate and Severe Anaemia in a
High-HIV and TB-Prevalent Adult
Population in the Eastern Cape
Province, South Africa. Int. J. Environ.
Res. Public Health 2023, 20, 3584.
https://doi.org/10.3390/
ijerph20043584
Academic Editor: Paul B. Tchounwou
Abstract: Background: Anaemia affects one in four adults in South Africa, with a higher preva-
lence in persons with HIV and tuberculosis. The aim of this study is to characterise the causes of
anaemia in primary care and a district hospital setting. Methods: A cross-sectional study design
investigated a purposive sample of adult males and non-pregnant females at two community health
centres and a hospital casualty and outpatients. Fingerpick blood haemoglobin was measured with
HemoCueHb201+. Those with moderate and severe anaemia underwent clinical examination and
laboratory tests. Results: Of 1327 patients screened, median age was 48 years, and 63.5% were female.
Of 471 (35.5%) with moderate and severe anaemia on HemoCue, 55.2% had HIV, 16.6% tuberculosis,
5.9% chronic kidney disease, 2.6% cancer, and 1.3% heart failure. Laboratory testing confirmed
227 (48.2%) with moderate and 111 (23.6%) with severe anaemia, of whom 72.3% had anaemia of
inflammation, 26.5% iron-deficiency anaemia, 6.1% folate deficiency, and 2.5% vitamin B12 deficiency.
Overall, 57.5% had two or more causes of anaemia. Multivariate modelling showed that patients with
severe anaemia were three times more likely to have tuberculosis (OR = 3.1, 95% CI = 1.5–6.5; p-value
= 0.002). Microcytosis was present in 40.5% with iron deficiency, macrocytosis in 22.2% with folate
deficiency, and 33.3% with vitamin B12 deficiency. The sensitivities of the reticulocyte haemoglobin
content and % hypochromic red blood cells in diagnosing iron deficiency were 34.7% and 29.7%,
respectively. Conclusions: HIV, iron deficiency, and tuberculosis were the most prevalent causes
of moderate and severe anaemia. The majority had multiple causes. Iron, folate, and vitamin B12
deficiencies should be identified by biochemical testing rather than by red cell volume.

Received: 8 December 2022 Keywords: anaemia causes; HIV; tuberculosis; nutritional deficiencies; anaemia of inflammation;
Revised: 13 February 2023 primary care
Accepted: 14 February 2023
Published: 17 February 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Anaemia is a significant public health problem worldwide, with adverse effects on
health and socio-economic development [1]. Global anaemia prevalence in 2019 was 23.2%,
affecting 1.8 billion people, resulting in an estimated 50.3 million years lived with disabil-
ity [2]. Dietary iron deficiency is the most prevalent cause globally, followed by inherited
hemoglobinopathies and haemolytic anaemias [2]. Iron deficiency (ID) is recognised as a
significant contributor to anaemia and ill-health in patients with common chronic inflamma-
tory conditions including chronic kidney disease (CKD) [3], heart failure [4], and cancer [5].
There is also evidence of inadequate investigation of anaemia in primary care [6–8].

Int. J. Environ. Res. Public Health 2023, 20, 3584. https://doi.org/10.3390/ijerph20043584 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2023, 20, 3584 2 of 17

Anaemia is common in South Africa, with community rates between 22% and 31% in
women and 12% and 17% in men older than 15 years [9,10]. In addition, South Africa has
8.2 million persons living with human immunodeficiency virus infection (PLWHIV) [11]
and an associated anaemia prevalence up to 72% [12]. South Africa also has a high tubercu-
losis (TB) prevalence estimated at 737 per 100,000 in 2018 [13]. Anaemia is a risk factor for
TB [12,14], and TB is associated with a high prevalence of anaemia (61.5%) [15].
ID is probably the most common cause in South Africa [9]. Anaemia of inflammation
(AI), which is synonymous with anaemia of chronic disease, is the second most common
cause globally in clinical practice [16] and, most probably, in South Africa [17,18]. In persons
living with HIV (PLWHIV), causes may be multifactorial, but anaemia of chronic disease is
the predominant cause [19]. In Southern Africa, TB is probably the most common cause of
moderate-to-severe anaemia in PLWHIV [12,20–22].
The relative contributions of all causes of anaemia are not well characterised in patients
attending primary care and district hospitals in South Africa. In a community-based study
in the Free State Province, AI was the most prevalent cause of anaemia in women with or
without HIV [17]; and in a district hospital (GF Jooste) in Cape Town, >95% of patients
with HIV-associated TB had AI and <3% had IDA [23]. However, no testing was done for
other causes of anaemia in either study.
The aims of this study were to determine the causes of anaemia in adults in primary
care and a district hospital setting in Mthatha, South Africa. To determine the overall causes
in a resource-efficient manner, the study focused on moderate and severe anaemia.

2. Materials and Methods

2.1. Study Design
A cross-sectional study design was used. The study was undertaken between Septem-
ber 2017 and March 2018.

2.2. Study Population and Setting

The study population was patients aged ≥18 years who attended two community
health centres (CHCs), one semi-urban (CHC1) and one rural (CHC2), and Mthatha Re-
gional Hospital (MRH) general outpatients and Emergency Department managed by the
Department of Family Medicine and Rural Health. MRH functions as both a district
and referral hospital. The catchment area is King Sabata Dalindyebo Local Municipality,
comprising a socio-economically deprived predominantly rural population [24].

2.3. Sampling
The population prevalence of moderate and severe anaemia was estimated to be
±2% [9]. This study was facility-based, and the prevalence was therefore estimated to
be higher at a minimum of 3%. Using the equation n = p (100 − p) (1.96)2 /d2 , where
n = minimum sample size, p = anticipated prevalence of moderate and severe anaemia,
and d = set precision [25]; the precision was set at 2% and the estimated prevalence at 3%,
yielding a minimum sample size of ±279 participants. The confidence interval was 95%. A
further 20% (56) of participants was added to the sample to factor in for anticipated loss
of participants during the evaluation process, loss of data, and data entry errors [26] for a
total sample size of 335 participants.

2.4. Study Procedures

A purposive sample was taken. Patients waiting to consult the nurse or doctor were
approached by a research assistant (a nurse at each site during normal working hours).
If agreeable, they signed a consent form. Exclusion criteria were: (1) pregnancy, (2) a
significant bleeding episode in the last three months that needed an urgent visit to the clinic
or doctor or a blood transfusion, and (3) unwillingness to disclose or test for HIV.
Entry to the study was by identifying moderate and severe anaemia using a HemoCue
Hb201+ analyser® on a capillary (finger-prick) sample. Anaemia was defined as mild,
Int. J. Environ. Res. Public Health 2023, 20, 3584 3 of 17

11–11.9 g/dL for women and 11–12.9 g/dL for men; moderate, 8–10.9 g/dL; and severe as
<8 g/dL for both sexes [27]. All patients had their HIV status assessed and a rapid HIV
test done if not on antiretroviral therapy (ART). ART was endorsed for all PLWHIV from
September 2016 [28]. Patients with mild anaemia were referred for routine care [29]. Those
with moderate and severe anaemia had the following tests analysed at the National Health
Laboratory Service (NHLS) at Nelson Mandela Central Hospital, Mthatha: full blood count,
reticulocyte production index (RPI), and blood film; creatinine, eGFR (Modification of
Diet in Renal Disease study equation, without the race coefficient), bilirubin (direct and
indirect), C-reactive protein, vitamin B12 and folate, iron, transferrin, transferrin saturation
(TSAT), and Xpert MTB/RIF test® on (non-induced) sputum. In addition, patients with
severe anaemia had urine and blood cultures for TB (BACTEC™ Myco/F Lytic Culture
vials) and urine lateral flow lipoarabinomannan assay LF-LAM (Alere® ). In PLWHIV, CD4
count and viral load (VL) tests were done according to national guidelines [28,30]. VL
suppression was defined as < 50 RNA copies/mL [30]. Patients were also assessed by a
doctor and underwent chest-X-ray and focused ultrasound examination for evidence of
TB [31] and chronic kidney disease (CKD). Additional tests were ordered at the treating
clinicians’ discretion. A final assessment/diagnosis was then made.
A Siemens ADVIA 2120 analyser was used initially followed by a Beckman Coulter LH
780 Analyser. The NHLS normal adult Hb values were 13–17 g/L for males and 12–15 g/L
for females. Microcytosis and macrocytosis were defined as MCV <80 fL and >100 fL,
respectively [32,33]. A % hypochromic red cells (%HRC) value >6% [34,35] or a reticulocyte
haemoglobin content (CHr) of <29 pg/cell indicate ID [35].
Vitamin B12 deficiency was categorised as: <147.6 pmol/L (200 pg/mL), deficiency likely;
>258.3 pmol/L (350 pg/mL), deficiency unlikely; while <73.8 pmol/L (100 pg/mL) usually in-
dicates clinical deficiency [36]. With vitamin B12 values between 147.6–258.3 pmol/L, patient
records were evaluated to determine if clinical findings were consistent with deficiency. A
serum folate level <6.8 nmol/L was defined as deficiency [37].
TB was diagnosed by detection of Mycobacterium tuberculosis (TB) by molecular testing
(Xpert MTB/RIF, Genotype® MTBDRsl), urine LF LAM, culture, or demonstration of
acid-fast bacilli on clinical specimens. Patients with probable TB diagnosed on X-ray or
ultrasound underwent anti-tuberculosis therapy but were excluded from analysis.
IDA was defined as anaemia and a ferritin < 30 mcg/L [38,39] and in the presence of
inflammation, a ferritin < 100 mcg/L, and ferritin 100–300 mcg/L if TSAT < 20% [39,40].
AI was diagnosed on three criteria: (1) a chronic inflammatory illness including cancer and
haematological malignancies, infections, immune-related diseases, inflammatory diseases,
CKD, heart failure, chronic pulmonary disease, and anaemia of the elderly [41]; (2) a ferritin
100–300 mcg and TSAT ≥ 20% or ferritin > 300 mcg/L [40]; and (3) the absence of an
identifiable cause [16]. Guidelines for heart failure [42], cancer [43], and CKD [44] accept
that inflammation is part of the disease process without a requirement for an elevated CRP.
A CRP (NHLS normal value < 10 mg/L) was performed primarily to identify inflammation
in people without a diagnosis of an inflammatory disorder.
CKD was diagnosed on standard criteria [45,46] and is considered the most likely
cause of anaemia if the GFR < 60 mL/min/1.72 m2 when no other cause is identified [44,47].

2.5. Statistical Analysis

Data were captured in Microsoft Excel and exported to STATA version 17 for analysis.
Numerical data were explored for normality using the Shapiro–Wilk test. All numerical data
were not normally distributed and were summarised using the median and interquartile
range (IQR = 75th percentile − (minus) 25th percentile). Categorical data were summarised
using percentages. A comparison of two categorical variables was undertaken using the
chi-square test if expected frequencies were ≥5, and if the expected frequencies were <5,
then the Fisher’s exact test was used. A comparison of medians used the Wilcoxon rank-
sum test for binary categorical variables and the Kruskal–Wallis test for nominal categorical
variables (more than two groups). Predictors of anaemia severity were determined using
Int. J. Environ. Res. Public Health 2023, 20, 3584 4 of 17

bivariable and multivariable logistic regression analysis. The adjusted multivariable logistic
regression model was determined through purposeful selection of variables to determine
the best-fitting model. The odds ratio or adjusted odds ratio (OR/aOR) was the measure of
association used for categorical predictors, and coefficients were used to predict numerical
variables. The 95% confidence interval (95%CI) was used for the precision of estimates,
and statistical significance is a p-value of <0.05. Because of the use of a subsample in the
analyses, especially for Tables 6 and 7, the p-values are purely descriptive. Missing data
were analysed using complete case analysis.

3. Results
A total of 21 declined participation in the study. The most common reason (5) was
unreadiness to know their HIV status. Table 1 summarises the demographic characteristics
of patients who participated, and it shows that 1327 participants were enrolled into the
study, of which 44.6% (592) were seen at MRH, and 63.5% (842) were female. The combined
median age was 48 years (IQR = 31 years). There was statistical homogeneity between
males and females (p-value = 0.100), the CD4 count (p-value = 0.539), and viral load
(p-value = 0.235). The median ages of patients were statistically different depending on
their recruiting health facility (p-value = 0.0003). Even though the combined HIV prevalence
was 20.9% (277/1327), it ranged from 5.5% (22/399) in CHC1 to 35.8% (212/592) at MRH,
and this was statistically significant (p-value < 0.0001). The HemoCue also showed anaemia
prevalences of 48.4% (147/304) and 46.1% (246/534) for males and females 40 years old
and older, respectively.

Table 1. Participant demographic characteristics and HemoCue Hb.

Characteristics MRH CHC1 CHC2 Total p-value

n = 592 n = 399 n = 336 n = 1327
Sex; n (%)
Female 362 (61.2) 251 (62.9) 229 (68.2) 842 (63.5)
Male 230 (38.9) 148 (37.1) 107 (31.9) 485 (36.6) 0.100 *
Age, years; med (p25–p75) 45 (31.5–60.0) 53 (37.0–67.0) 48.5 (34.0–64.0) 48 (33.0–64.0) 0.0003 U *
Age categories, years; n (%)
18–29 101 (18.8) 58 (14.5) 50 (14.9) 219 (16.5)
30–39 131 (22.1) 55 (13.8) 70 (20.8) 256 (19.3)
40–59 188 (31.8) 147 (36.8) 107 (31.9) 442 (33.3)
60–74 103 (17.4) 82 (20.6) 68 (20.2) 253 (19.1) 0.011 *
≥75 59 (10.0) 57 (14.3) 41 (12.2) 157 (11.8)
HIV; n (%)
Positive 212 (35.8) 22 (5.5) 43 (12.8) 277 (20.9)
Negative 380 (64.2) 377 (94.5) 293 (87.2) 1050 (79.1) <0.0001 *
CD4, cells/µL; med (p25–p75) 101.5 (38–246.0) 48.5 (15.5–413.0) 150 (45–246.5) 101.5 (36–252.0) 0.539 U *
VL, log(copies/mL); med
3.6 (1.8–5.7) 4.2 (2.4–5.9) 4.8 (3.0–5.7) 3.8 (1.9–5.7) 0.235 U *
(p25–p75)
Anaemia, HemoCue; n (%)
Normal 168 (28.3) 292 (73.2) 224 (66.7) 684 (51.5)
Mild 86 (14.5) 45 (11.3) 41 (12.2) 172 (13.0)
Moderate 190 (32.0) 50 (12.5) 50 (14.9) 290 (21.9) <0.0001 *
Severe 148 (25.0) 12 (3.0) 21 (6.3) 181 (13.6)
Hb-HemoCue, g/dL; med
10 (8.0–12.5) 13 (12.0–14.1) 12.5 (11.2–13.5) 12 (9.6–13.5) 0.0001 U *
(p25–p75)
p75, 75th percentile minus; p25, 25th percentile; med, median; VL, viral load; Hb, haemoglobin; U * * Chi-squared
test was used; Kruskal–Wallis test was used.

Of the 1327 patients, 471 (35.5%) qualified to have a laboratory evaluation of their
anaemia due to HemoCue blood levels of moderate or severe anaemia status (Table 2).
However, only 463/471 (98.3%) participants had laboratory haemoglobin results, of whom
49.0% (n = 227) had moderate and 24.0% (n = 111) severe anaemia, comprising 25.5%
(n = 338) of those screened. Chronic diseases that can cause anaemia (HIV, TB, CKD, can-
cer, and heart failure) and non-communicable diseases (NCDs) of high prevalence were
assessed on the 471 participants who had progressed for a laboratory evaluation. The
median haemoglobin levels were statistically different between the three health facilities
(p-value = 0.002), and the difference was due to the lower median of MRH (9.4) when
compared to CHC1 (10.9), which was statistically significant (p-value < 0.001). Disease
prevalences were as follows: tuberculosis (16.6%, n = 78), hypertension (20.0%, n = 94),
 Int. J. Environ. Res. Public Health 2023, 20, 3584 5 of 17

chronic kidney disease (5.9%, 28), and cancers (2.6%, n = 12). There was a statistically signif-
icant difference in the hypertension status depending on the health facility of recruitment
(p-value < 0.001). Of those with confirmed TB, 54/78 (69.2%) had pulmonary TB, 13/78
(16.7%) extra-pulmonary TB, 7/78 (9.0%) had both, and in 4/78 (5.1%), the primary site
could not be confirmed. In addition to TB, there were only two patients with CDC-Stage-3-
defining illnesses [48], namely cervical cancer and cryptococcal antigenaemia. More than
two-thirds of the participants with HemoCue severe and moderate anaemia who had HIV
were in CDC Stage 3. The CD4 nadir, median, mean, and zenith were 1, 103, 191, and 2440,
respectively. Viral replication was not suppressed in the majority of PLWHIV.

Table 2. Clinical conditions of participants with haemoglobin <11 g/dL by HemoCue.

Characteristics MRH CHC1 CHC2 Total p-Value

n = 338 n = 62 n = 71 n = 471
Laboratory Hb **, g/dL;
9.4 (7.8–10.9) 10.9 (9.5–11.9) 9.7 (8.1–10.7) 9.7 (8.0–11.1) <0.001 U *
med (p25–p75)
Laboratory Hb **; n (%)
Normal 32 (9.5) 13 (21.0) 7 (9.9) 52 (11.0) 0.002 *
Mild 49 (14.5) 17 (27.4) 7 (9.9) 73 (15.5)
Moderate 6 of16318 (48.2) 25 (40.3) 39 (54.9) 227 (48.2)
Severe 89 (26.3) 6 (9.7) 16 (22.5) 111 (23.6)
Missing 5 (1.5) 1 (1.6) 2 (2.8) 8 (1.7)
Vitamin B12 = , pmol/L;
562 (360.0–1051.0) 383 (280.0–709.0) 425.5 (292.0–568.0) 492 (333.0–943.0) <0.001 U *
med (p25–p75)
ж
157 (82.6)
Folate , nmol/L; med
18.4 (11.3–27.5) 18.7 (11.3–31.9) 23.5 (15.8–32.7) 19.1 (12.0–29.7) 0.019 U *
(p25–p75) ʊ
)ic Health
0.62021,Transferrin
(0.3–0.9)
18, x FOR PEER REVIEW 16.0
saturation ß0.003 *
*, 6 of 18
(10.0–28.0) 17 (12.0–21.0) 13 (7.0–19.0) 16 (10.0–25.0) 0.024 U *
7) 32 %; med (p25–p75) 0.016 ʊ*
(30.7–33.1)
Ferritin = , µg/L; med (139.0–
678.5 (222.0–1569.0) 177.5 (69.0–520.0) 154 (49.0–1686.0) 520 <0.001 U *
(p25–p75) 14206.0)
45 (9.6)
HIV; n (%)
mL 423 120 Positive (80.5)0.370 * 201 12 (59.5)
(92.3) 25 18 (89.3) (29.0) 157 41(82.6) (57.8) 260 (55.2)
(90.4)
Negative 137 (40.5) 44 (71.0) 30 (42.3) 211 (44.8) <0.001 *
p25–p75) CD40.5 ) (0.3–0.9) 0.7 (0.5–1) 0.6 (0.3–0.8) 0.6 (0.3–0.9) 0.003 ʊ*
*, cells/µL; med
100 (37–252) 67.5 (21–413) 150 (43–242) 103 (36–255) 0.866
(p25–p75)
94 32.2 (p25–p75)
(20.0)
)
(30.7–33.3) 31.8 (30.9–32.9) 31.8 (30.3–32.7) 32 (30.7–33.1) 0.016 ʊ*
CD4 *, cells/µL; n (%) <0.001 *
ȸ; n (%)
376 (80.0)
<200 (Stage 3) 125 (67.6) 10 (62.5) 21 (61.8) 156 (66.4)
200–499
29 (Stage(8.7) 2) 42
6 (22.7)
(9.7) 10 3 (14.1) (18.8) 45 11 (9.6) (32.4) 56 (23.8) 0.492 ***
≥500 (Stage 1) 18 (9.7) 3 (18.8) 2 (5.9) 0.370 * 23 (9.8)
78 306
(16.6)
Viral load; n (%) (91.3) 56 (90.3) 61 (85.9) 423 (90.4)
; n (%)277 <50 copies/mL 29 (19.5) 1 (7.7) 3 (10.7) 33 (17.4) 0.455 ***
(58.8)
≥50 copies/mL 0.051 ***120 (80.5) 12 (92.3) 25 (89.3) 157 (82.6)
7 49
(1.5)med (p25–p75)
RPI, count; (14.5) 29
0.5 (46.8)
(0.3–0.9) 16 0.7 (22.5) (0.5–1) 94 0.6(20.0) (0.3–0.8) 0.6 (0.3–0.9) 0.003 U *
MCHC, g/dL; (85.5)
med <0.001 *
109 288
(23.1) 33
32.2 (53.2)
(30.7–33.3) 55 31.8 (77.5) 376
(30.9–32.9) (80.0)
31.8 (30.3–32.7) 32 (30.7–33.1) 0.016 U *
(p25–p75)
n (%) Diabetes mellitus ) ; n (%)
58 Yes 29 (8.7) 6 (9.7) 10(16.6) (14.1) 45 (9.6)
28 (5.9)No (17.2) 7
306
(11.3)
(91.3)
13
56
(18.3)
(90.3)
78
61 (85.9) 423 (90.4) 0.370 *
193 # (57.1) 0.703 ***36 (58.1) 48 (67.6) 277 (58.8)
443 (94.1) ; n (%)
Hypertension
ted 3 Yes (0.9) 49
2 (14.5)
(3.2) 2 29 (2.8) (46.8) 7 16 (1.5) (22.5)0.051 *** 94 (20.0)
No 288 (85.5) 33 (53.2) 55 (77.5) 376 (80.0) <0.001 *
¶
12 84
Tuberculosis;
(2.6) n(24.9)
(%) 17 (27.4) 8 (11.3) 109 (23.1)
ease; 455
n (%) Yes 1.000 ***58 (17.2) 7 (11.3) 13 (18.3) 78 (16.6)
(96.8) No 193 (57.1) 36 (58.1) 48 (67.6) 277 (58.8)
22 0.051 ***
3 (0.6) ¶ (6.5)
Contaminated 33 (4.8)(0.9) 3 2 (4.2) (3.2) 28 2 (5.9) (2.8) 7 (1.5)
Missing 84 (24.9) 17 (27.4) 8 (11.3)0.703 *** 109 (23.1)
316
Chronic kidney disease; n
(93.5) 59 (95.2) 68 (95.8) 443 (94.1)
(%) 6 (1.3)(%)
464 9 Yes
(98.7) No
(2.7)0.293 ***316 22
1 (1.6)(6.5)
(93.5)
2 3
59
(2.8) (4.8)
(95.2)
12 3
68
(2.6) (4.2)
(95.8)1.000
28
443
(5.9)
(94.1) 0.703 ***
***
ж missing = 69; 325 ȹ
Cancer
missing
# (96.4)
; n (%)= 35; 61
ß* missing (98.4) 69 (97.2) 455 (96.8)
Yes ˆ
Ҧ
cancer = 2; cervix, 3 squamous
No
(0.9) carcinoma 09
325
(2.7)
(0)(96.4) 0 1
61
(0) (1.6)
(98.4)
3 2
69
(0.6) (2.8)
(97.2)
12
455
(2.6)
(96.8)
1.000 ***
n (%)
myeloid leukaemia= Probable 1; Ҧ probable bone 3 (0.9) 0 (0) 0 (0) 3 (0.6)
#
ma = 1; ¶ excludes Heart failurewith
4those ; n(1.2)
(%)probable 2TB
(1.7) 0 (0) 6 (1.3)
Yes (1.2) 4 2 (1.7) 0 (0) 6 (1.3)
333 NoHb,
rcentile; med, median; (98.8) (96.8)
haemoglobin.
(98.8)60
333 71 60 (100.0) (96.8) 464 71(98.7) 0.293 *** 464
(100.0) (98.7) 0.293 ***
d; ʊ* Kruskal–Wallis
# missingtest
= 1;was used. = 8; ȸ #missing = 3; ȸ* missing = 25;
** missing )
ж missing = 69; ȹ missing = 35; ß* missing
) ж = ß
missing = 1; ** missing = 8; missing = 3; * missing = 25; missing = 69; missing = 35; * missing = 78;
= 78; ^ Prostate cancer = 3; oesophageal cancer
ˆ Prostate cancer = 3;and breast cancer
oesophageal cancer and= 2;breast
cervix, squamous
cancer carcinoma
= 2; cervix, squamous carcinoma neck, adenocarcinoma
of IDA andneck, adenocarcinoma
AI. The category AIlung,in- multiple myeloma,
lung, multiple andand
myeloma, acute myeloid
acute myeloidleukaemia=
leukaemia= 1; 1; Ҧ probable bonecancer, undetermined pelvic mass,
probable bone
cancer,and
undetermined and probable
pelvic mass, lymphoma
and probable = 1; ¶ excludes
lymphoma = 1; ¶ those with those
excludes probable
withTBprobable
based on X-ray
TB diagnosis. p75, 75th percentile;
amin deficiencies haematological
p25, 25th percentile; med, median; Hb, haemoglobin. * Chi-squared test was used; *** Fisher’s exact test was used;
based on
g 17 with missing X-ray diagnosis.
ferritin p75,U75th percentile; p25, 25th percentile; med, median; Hb, haemoglobin.
results, the * Kruskal–Wallis test was used.
* Chi-squared test was used; *** Fisher’s exact test was used; ʊ* Kruskal–Wallis test was used.
d 26.5% (85/321), respectively. In pa-
nts with cancer, 1/6 (16.7%) had IDA.
Figure 1 illustrates the biochemical diagnosis of IDA and AI. The category AI in-
cludes those with specific causes of anaemia (e.g., vitamin deficiencies and haematological
malignancies) who also had inflammation. Excluding 17 with missing ferritin results, the
percentages of AI and IDA were 72.3% (232/321) and 26.5% (85/321), respectively. In pa-
tients with CKD, 8/25 (32%) had IDA. Of nine patients with cancer, 1/6 (16.7%) had IDA.
Int. J. Environ. Res. Public Health 2023, 20, 3584 6 of 17

Figure 1 illustrates the biochemical diagnosis of IDA and AI. The category AI includes
those with specific causes of anaemia (e.g., vitamin deficiencies and haematological ma-
lignancies) who also had inflammation. Excluding 17 with missing ferritin results, the
percentages of AI and IDA were 72.3% (232/321) and 26.5% (85/321), respectively. In
Int. J. Environ. Res. Public Health 2021,patients with
18, x FOR PEER CKD, 8/25 (32%) had IDA. Of nine patients with cancer,
REVIEW 7 of1/6
18 (16.7%) had IDA.
Four patients with cardiac failure had AI.

Figure
Figure1.1.
Diagnosis of iron-deficiency
Diagnosis anaemia and
of iron-deficiency anaemiaand
anaemia of inflammation.
anaemia ofCappellini et al. [40]Cappellini
inflammation. et al. [40]
and Camaschella and Girelli, 2020 [39].
and Camaschella and Girelli, 2020 [39].
Only 399 individuals had a full record of the time from venesection to FBC analysis.
Only
The time 399 individuals
a specimen was receivedhad a full
at the recordwas
laboratory of used
the time fromforvenesection
as a proxy the time to to FBC analysis.
The time
analysis, a specimen
as reports did not was received
specify at the
analysis times. laboratory
With a median ofwas
2.6 hused
(IQR =as2.2ah),
proxy
all for the time to
FBC analyses were undertaken within 24 h. Figure 2 shows no statistically significant
analysis, as reports did not specify analysis times. With a median of 2.6 h (IQR = 2.2 h), dif-
ference in the median MCV in those analysed < 8 h and ≥ 8 h (p = 0.766) and in the median
all FBC analyses were undertaken within 24 h. Figure 2 shows no statistically significant
%HPO for those analysed < 6 h and ≥ 6 h (p = 0.176).
difference in the median MCV in those analysed <8 h and ≥8 h (p = 0.766) and in the
median %HPO for those analysed <6 h and ≥6 h (p = 0.176).
Using RHC to diagnose IDA, as shown in Table 3, sensitivity was 34.7%, specificity
89.4%, and positive predictive value 64.7%.
Table 4 shows the use of HRC to diagnose IDA. Sensitivity was 29.7%, specificity
87.9%, and positive predictive value 92.2%.
Table 5 shows that patients with ID had predominantly normocytic anaemia, as
also did patients with folate deficiency. Patients with vitamin B12 deficiency had equal
proportions of microcytic, normocytic, and macrocytic anaemia. In patients 65 years old
and older with IDA, 35.3% (6/17) had microcytosis.
In eight patients with vitamin B12 deficiency, two had neurological disorders consistent
with deficiency. One had acute confusion (vitamin B12, 50 pmol/L) and one lumbar
radiculopathy (vitamin B12, 75 pmol/L). They had normal MCV and no blood film features
of megaloblastic anaemia. Another patient was on metformin, which may cause deficiency.
Five had no neurological or haematological features to diagnose deficiency. Of three
patients with possible vitamin B12 deficiency (range 147.6–258.3 pmol/L), one had acute
psychosis with HIV and hepatitis B virus infections, one had paraparesis of unknown
Int. J. Environ. Res. Public Health 2023, 20, 3584 7 of 17

cause, and one PLWHIV had TB meningitis. Overall, 2.5% (8/324 results) had vitamin
Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 8 of 18
B12 deficiency.

Figure 2. Duration of time to analysis of blood specimen for MCV and HPO.
Figure 2. Duration of time to analysis of blood specimen for MCV and HPO.
Using RHC to diagnose IDA, as shown in Table 3, sensitivity was 34.7%, specificity
89.4%, and positive predictive value 64.7%.
Table 3. Iron-Deficiency Anaemia and Reticulocyte Haemoglobin Content.
Table 3. Iron-Deficiency Anaemia and Reticulocyte Haemoglobin Content.
RHC < 29 RHC > −29 Total
Iron-Deficiency
Iron-Deficiency Anaemia Anae- RHC < 29 RHC > −29 Total
mia n n % % n n % % N N% %
Yes Yes 33
33 (34.7)
(34.7) 18
18 (10.6)
(10.6)51 (19.2)
51 (19.2)
No 62 (65.3) 152 (89.4) 214 (80.9)
No Total 95 62 (65.3)
(100.0) 170 152 (100) (89.4)265 214
(100) (80.9)
Total 95 (100.0) 170 (100) 265 (100)
Table 4 shows the use of HRC to diagnose IDA. Sensitivity was 29.7%, specificity
87.9%, and positive predictive value 92.2%.
Table 4. Iron-Deficiency Anaemia and % Hypochromic Red Cells.
Table 4. Iron-Deficiency Anaemia and % Hypochromic Red Cells.

Iron-Deficiency Anae- %HPO Analysis

%HPO Analysis Total Total
Iron-Deficiency Anaemia
mia ≥6% <6%
Yes n (%) 47
≥ 6%
(92.2) 4
<6%
(7.8) 51 (100.0)
No; n (%)
Yes n (%) 11147 (79.3)
(92.2) 29 4 (20.7) (7.8)
140 (100.0)
51 (100.0)
Total; n (%) 158 (82.7) 33 (17.3) 191 (100.0)
No; n (%) 111 (79.3) 29 (20.7) 140 (100.0)
Total;Table
n (%)5 shows that patients158
with ID had predominantly
(82.7) 33 normocytic anaemia,191
(17.3) as also (100.0)
did patients with folate deficiency. Patients with vitamin B12 deficiency had equal pro-
portions of microcytic, normocytic, and macrocytic anaemia. In patients 65 years old and
Table 5. Iron,older with IDA,
Folate, and 35.3% (6/17)B12
Vitamin had deficiency
microcytosis.and Mean Corpuscular Volume in laboratory
moderate/severe
Tableanaemia.
5. Iron, Folate, and Vitamin B12 deficiency and Mean Corpuscular Volume in laboratory mod-
erate/severe anaemia.
Deficiency Microcytic Normocytic Macrocytic TOTALS
Iron; * n (%) 34 (40.5) 47 (56.0) 3 (3.6) 84 (100.0)
Folate; n (%) 3 (16.7) 11 (61.1) 4 (22.2) 18 (100)
Vitamin B12; n (%) 3 (33) 3 (33) 2 (33) 8 (100.0)
Iron-deficiency anaemia: ferritin <30 mcg/L and with inflammation, <100 mcg/L, or 100–300 mcg/L plus
TSAT < 20% [39]. A folate level <6.8 nmol/L defined deficiency [37]. Vitamin B12 deficiency was defined as
<147.6 pmol/L (200 pg/mL) [36]. * One patient with IDA had a missing MCV result.

In patients with moderate and severe anaemias and a vitamin B12 level <147.6 pmol/L,
none had hyper-segmented neutrophils or Howell–Jolly bodies. One patient had macrocy-
tosis and ovalocytes with a vitamin B12 level of 109 pmol/L and a folate level of 2.1 pmol/L.
Int. J. Environ. Res. Public Health 2023, 20, 3584 8 of 17

The prevalence of folate deficiency was 6.1% (18/294 results). None of the patients with
folate deficiency were taking a medication causing deficiency.

3.1. Macrocytosis
Of 25 patients with macrocytosis, 10 had incomplete drug data and 15 were PLWHIV.
The putative causes of macrocytosis in 15 were lamivudine, 8; folate deficiency, 3; vitamin
B12 deficiency, 2; cotrimoxazole, 2; valproate, 2; zidovudine, 2; liver disease, 1; and multiple
myeloma, 1 (four patients had two causes each, and one had three).

3.2. Microcytosis
Of 78 patients with microcytosis, one had a Mentzer index value < 13. That patient
had IDA (TSAT 4%, ferritin 46 mcg/L).

3.3. Multiple Causes of Anaemia

For each of the eight major causes, the numbers (percentage) of patients with mod-
erate/severe anaemia due solely to that cause were iron deficiency 32/85 (37.6), vitamin
B12 deficiency 4/8 (50), folate 1/18 (5.6), HIV 136/219 (62.1), cancer 2/12 (16.7), CKD 8/28
(28.6), TB 6/75 (8.0), and heart failure 2/4 (50). Overall, 191/449 (42.5%) had a single cause
of anaemia, ranging from 5.6% for patients with folate deficiency to 62.1% with HIV. The
remainder (57.5%) had two or more causes.
In the bivariable model, those who had severe anaemia were 70% (OR = 1.7,
95%CI = 1.0–2.7; p-value = 0.039) and 90% (OR = 1.9, 95%CI = 1.1–3.2; p-value = 0.017)
more likely to have HIV and TB, respectively (Table 6). However, the multivariable best-
fitting model that was adjusted for other chronic conditions and the anaemia markers
did not show an association between the severity of anaemia and an HIV-positive result
(p-value = 0.880). Instead, the measure of effect increased for TB, wherein patients with
severe anaemia were found to have been three times more likely to have had TB, and
this was also statistically significant (OR = 3.1, 95% CI = 1.5–6.5; p-value = 0.002). Sim-
ilarly, patients with severe anaemia were 5.5 times more likely to present in a hospital
setting than a primary care setting, and this was statistically significant as well (OR = 5.5,
95%CI = 1.5–19.7; p-value = 0.009).
The multivariable analysis further shows that an increase of 1 in the reticulocyte pro-
duction index increased the odds of severe anaemia by 5.5 (co-efficient = 1.7,
95%CI = 0.9–2.5; p-value < 0.0001); and a 1 g/dL increase in the mean corpuscular
haemoglobin concentration (MCHC) increases the odds of having severe anaemia by
65% (co-efficient = 0.5, 95%CI = 0.4–0.7; p-value < 0.0001), and both these were statisti-
cally significant.
Even though PLWHIV with moderate anaemia had a statistically lower median Hb
than HIV-negative patients (p-value < 0.0004), there was no statistical difference between
the median Hb of patients with a negative HIV status and PLWHIV in patients with
severe anaemia (p-value = 0.623) (Table 7). TB showed an opposite effect where pa-
tients with severe anaemia had a higher median Hb, and this was statistically significant
(p-value = 0.005). The median MCHC was lower than normal in patients with moderate
and severe anaemia and lower still in patients with severe anaemia and HIV or TB. Other
markers of statistical significance included the following: a higher median CRP for PLWHIV
with moderate anaemia (p-value = 0.003); higher median CRP values for both TB-positive
patients with moderate (median = 146; p-value = 0.005) and severe anaemia (median = 163;
p-value = 0.0004); higher median transferrin saturation severe anaemia in both HIV-positive
(median = 19.6%; p-value = 0.0004) and TB-positive patients (median = 19.2%; p-value = 0.015);
lower median RPIs for PLWHIV with moderate (median = 0.5; p-value = 0.005) and severe
anaemia (median = 0.3; p-value = 0.036); and higher median ferritin levels for all categories
of HIV-positive and TB-positive patients (p-value < 0.05). Vitamin B12 values were higher
in PLWHIV with moderate or severe anaemia and in TB with moderate anaemia. Folate
levels were unchanged with HIV and TB.
Int. J. Environ. Res. Public Health 2023, 20, 3584 9 of 17

Table 6. Bivariable and Multivariable predictors of severe anaemia among patients with moderate or
severe anaemia.

Bivariable Analysis Multivariable Analysis

Variable
n (N = 338 ˆ *) OR/ß 95% CI p-Value aOR/ß 95% CI p-Value
HIV
Negative 33/119 ref - 1 ref - 1
Positive 78/219 1.7 (1.0–2.7) 0.036 0.9 (0.4–2.0) 0.880
TB
No 66/219 ref - 1 ref - 1
Yes 34/75 1.9 (1.1–3.2) 0.017 3.1 (1.5–6.5) 0.002
Cancer
No 107/322 ref - 1 ref - 1
Yes 4/12 1.1 (0.3–3.8) 0.871 3.9 (0.6–24.7) 0.153
Chronic kidney disease
No 102/310 ref - 1 ref - 1
Yes 9/28 1.1 (0.5–2.4) 0.878 0.8 (0.3–2.7) 0.763
Health facility
CHC1 6/31 ref - 1 ref - 1
CHC2 16/55 2.1 (0.7–5.9) 0.323 2.0 (0.5–8.2) 0.324
MRH 89/252 2.9 (1.2–7.1) 0.022 5.5 (1.5–19.7) 0.009
Sex
Female 73/229 ref - 1 ref - 1
Male 38/109 1.2 (0.7–1.9) 0.481 1.1 (0.5–2.2) 0.789
MCHC ß 7/168 0.3 (0.2–0.4) <0.0001 0.5 (0.4–0.7) <0.0001
RPI ß 6/154 1.3 (0.6–1.9) <0.0001 1.7 (0.9–2.5) <0.0001
Vitamin B12 * 1000 ß 7/164 −0.4 (−0.8; −0.04) 0.030 −0.7 (−1.2; −0.07) 0.028
Transferrin saturation * 20 ß 7/147 0.001 (−0.02; 0.004) 0.645 - - -
C-reactive protein * 100 ß 6/161 0.01 (−0.2; 0.2) 0.901 - - -
MCV ß 7/169 0.04 (0.02–0.1) 0.001 - - -
Ferritin * 10,000 ß 7/164 −0.01 (−0.5; 0.5) 0.966 - - -
Platelets * 100 ß 7/171 −0.05 (−0.2; 0.1) 0.457 - - -

OR, odds ratio; aOR, adjusted odds ratio; ˆ * complete cases in multivariable model = 266; ß coefficients used; ref,
reference category; 95%CI, 95% confidence interval; MRH, Mthatha Regional Hospital; n, number of patients with
severe anaemia; N, Total number of patients with severe and moderate anaemia.

Table 7. Median and interquartile range (IQR) comparison by HIV and TB status for participants
with laboratory severe and moderate anaemia.

HIV Tuberculosis

Moderate Severe Moderate Severe

Variable
HIV HIV HIV HIV p-
p-Value TB Positive TB Negative p-Value TB Positive TB Negative p-Value
Positive Negative Positive Negative Value

Hb, g/dL; med (IQR) 9.4 (1.4) 9.9 (1.3) 0.0004 6.7 (1.9) 6.4 (2.0) 0.623 9.7 (1.5) 9.5 (1.4) 0.939 7.1 (1.7) 6.1 (2.0) 0.005

MCV, fL; med (IQR) 88.9 (11.2) 87.9 (11.3) 0.610 86.2 (14.8) 83.5 (18.6) 0.279 86.1 (7.8) 88.6 (11.8) 0.156 85.7 (14.7) 85.4 (18.3) 0.963

MCHC, g/dL; med (IQR) 31.7 (2.3) 31.2 (1.9) 0.080 30.5 (2.2) 29.2 (4.7) 0.016 31.9 (2.1) 31.5 (2.2) 0.561 31.1 (2.6) 29.9 (3.4) 0.003

HPO, %; med (IQR) 15.8 (21.0) 20.6 (31.2) 0.063 28.9 (41.3) 68.4 (33.3) 0.010 10.6 (16.3) 20.2 (29.8) 0.142 31.9 (40.2) 59.1 (52.0) 0.031

RHC, pg; med (IQR) 31.3 (6.9) 30.8 (4.0) 0.524 31.0 (6.3) 25.2 (7.3) 0.004 30.2 (5.3) 31.0 (5.6) 0.673 29.9 (4.9) 29.4 (9.8) 0.297

RPI, count; med (IQR) 0.5 (0.6) 0.7 (0.5) 0.005 0.3 (0.3) 0.4 (0.5) 0.036 0.6 (0.6) 0.5 (0.5) 0.127 0.3 (0.3) 0.4 (0.3) 0.146

Platelets, * 109 /L; med (IQR) 307.0 (182) 331 (211) 0.110 294.0 (200) 355.0 (225.0) 0.051 357.0 (227) 314.5 (189.5) 0.483 252.0 (202.0) 330.0 (201.0) 0.059

Iron, µg/dL; med (IQR) 305.1 (299.3) 351.1 (236.0) 0.186 284.9 (316.6) 189.9 (184.2) 0.008 293.5 (184.2) 345.3 (316.6) 0.133 290.7 (293.5) 207.2 (241.7) 0.032

Transferrin, mg/dL; med

1380 (790) 1920 (1080) 0.0001 1305 (850) 1560 (1150) 0.010 1350 (570) 1685 (1070) <0.012 1160 (580) 1475 (1320) 0.009
(IQR)

Transferrin saturation (%);

16.5 (18.2) 14.5 (11.3) 0.171 19.6 (21.0) 7.3 (15.5) 0.0004 16.5 (9.3) 14.8 (15.3) 0.858 19.2 (20.0) 12.2 (19.6) 0.015
med (IQR)

712.0 1376.0 458.0 2000.0 550.0

Ferritin, µg/L; med (IQR) 423 (880.5) 0.003 162.0 (912.0) 0.0001 925 (1390.0) 0.0004 0.0001
(1590.0) (1771.0) (1153.0) (1751.0) (1349.0)

CRP, mg/L; med (IQR) 103.5 (198.0) 41.5 (129.0) 0.003 109.5 (148.0) 92.0 (143.0) 0.462 146.0 (214) 59.0 (162.0) 0.005 163.0 (122.0) 86.5 (132.5) 0.0004

Vitamin B12, pmol/L; med 785.0

522.5 (735.5) 416.0 (528) 0.009 767.5 (957.0) 530.0 (450.0) 0.043 688.0 (660.0) 445.0 (587.0) 0.002 595.0 (544.0) 0.129
(IQR) (1193.0)

Folate; nmol/L; med (IQR) 18.6 (17.8) 21.5 (14.8) 0.081 16.7 (16.0) 18.9 (23.9) 0.103 18.2 (16.9) 21.1 (16.9) 0.150 17.5 (15.3) 18.9 (23.6) 0.631

CD4, cells/µL; med (IQR) 103.5 (213.0) - - 72.0 (189.0) - - 68 (200.0) 114.0 (213.0) 0.178 47.0 (98) 124.0 (193.0) 0.013

VL, log(copies/mL); med

4.0 (3.7) - - 3.8 (3.3) - - 4.3 (3.2) 4.0 (3.8) 0.242 5.3 (3.2) 2.9 (2.7) 0.001
(IQR)

IQR, 75th percentile minus (−) 25th percentile; dL, decilitre; RHC, reticulocyte haemoglobin content; RPI,
reticulocyte production index. The National Health Laboratory Service in South Africa reports iron in µmol/L
and transferrin in g/L. Normal ranges: MCHC, males 33–35, females 32.7–34.9 g/dL; Iron-Deficiency Indices:
%HYPO > 6%; RHC < 29 pg/cell. The Wilcoxon rank-sum test was used.
Int. J. Environ. Res. Public Health 2023, 20, 3584 10 of 17

4. Discussion
There was an overall high (45.9%) prevalence of anaemia in the screened popula-
tion, with the highest prevalence at MRH. Similar to attendees in primary care in South
Africa [49], two-thirds of patients screened for anaemia in this study were female. In this
study, the age group 40–49 years had the largest number screened. This may be due to
the exclusion of pregnant women and the inclusion of sicker patients attending MRH. In
older adults (≥40 years old), 48.4% of men and 46.1% of women had anaemia. High rates
of anaemia were also found in community-living older adults in Mpumalanga Province,
South Africa, i.e., 40.1% of men and 43% of women [50]. Prevalence of HIV infection was
similar, at 20.9% in this study and 20.7% in Mpumalanga Province.
Overall, the largest category of anaemia on screening was moderate, followed by
severe. In contrast, in the national community surveys, the largest category was mild
followed by moderate [9,10]. The skewed distribution in this study is most likely due
to selection of participants from health care facilities, which will likely have more sicker
individuals than those in the general population. The overall prevalence of moderate and
severe anaemia was 35.5% on HemoCue testing but 25.5% on laboratory testing. HemoCue
201 overestimates mean Hb concentrations by 0.1–1.2 g/dL [51]. Furthermore, capillary
finger-prick usually produces higher Hb values by 0.2–0.9 g/dL compared to venous
blood [51].
In patients with laboratory-confirmed moderate and severe anaemia, AI was the
predominant type at 72.6%, reflecting the high prevalences of HIV (59.5%) and TB (16.6%).
In Cape Town, >95% of patients with anaemia and HIV-associated TB had AI [23]. In
patient populations where there are very low levels of HIV and TB diagnoses, AI was
the commonest type of anaemia, but prevalences were much lower at 25.7% in elderly
inpatients in South Africa [18] and 41.9% of medical inpatients in Italy [52]. In the U.S., one-
third of elderly community-dwellers with anaemia had AI, one-third nutrient deficiencies,
and one-third unexplained anaemia [53].
The 26.5% prevalence of IDA is similar to prevalences of 24.3% in older inpatients in
South Africa [18] and 20% in older community-dwellers in the U.S. [53] but higher than
14.7% in internal medicine inpatients in Italy [52]. IDA in this study comprised 15.9%, with
absolute ID (ferritin < 100 mcg/L [54]) and 10.5% functional ID (ferritin ≥ 100–300 mcg/L
and TSAT < 20%) [39,43]. However, absolute and functional ID overlap such that patients
who have ferritin > 100 mcg/L may have absolute iron deficiency. In a systematic review
utilising 38 studies, the mean ferritin level in absolute iron deficiency was 82.4 mcg/L
(range of means 34–158 mcg/L) in patients with inflammatory diseases, using bone marrow
iron as the gold standard [55]. While there are various definitions of ID [23], the “pragmatic”
definition of Camaschella and Girelli [39] was used in this study, combining ferritin and
TSAT. TSAT is considered an accurate test for ID in patients with inflammation [39,56].
Newer tests for ID include hepcidin and soluble transferrin receptor (sTfR) levels [40] but
are not routinely available in South Africa.
Forty-one per cent (41%) of patients with IDA had microcytosis. In patients 65 years old
and older, 35.3% had microcytosis. Studies in older patients with absolute IDA in developed
countries show <30% have microcytosis [57]. These data strongly support biochemical
testing for ID in all patients with anaemia irrespective of MCV values. Only one with
microcytosis had a Mentzer index value < 13 [58] but had concomitant ID. This suggests
that the thalassaemia trait is uncommon in the study population. While the α-thalassaemia
trait is present in some communities in South Africa, with a prevalence of 3.8% [59]
and 16% in non-random samples [60], testing is mainly done if there is unexplained
microcytosis [60,61].
All MCV measurements in the laboratory were done within 24 h of venesection, with
no difference between those measured before and after eight hours. While MCV stored
at 4 ◦ C remains unchanged for 24 h, it increases significantly after eight hours at room
temperature (standard mean difference −0.30, CI −0.50, −0.10)) [62].
Int. J. Environ. Res. Public Health 2023, 20, 3584 11 of 17

RHC and %HYPO had low sensitivity for IDA, implying that the tests are not suitable
for ruling out IDA in the study setting. RHC can perform as well as standard tests for
the diagnosis of ID [35]. %HPO has mainly been used to diagnose ID in the setting of
CKD [35,39]. While %HPO should be analysed within six hours of venesection [34], Figure 2
shows that there was no significant difference between blood samples drawn before or
after six hours. Further research is needed into the sub-optimal performance of these tests
in similar settings to this study.
The major disease categories associated with moderate and severe anaemia were, in
order, HIV, TB, CKD, and cancer, as shown in Table 2. Anaemia is common in PLWHIV from
multifactorial causes [63]. While worldwide prevalences in PLWHIV are 21.6%, 22.6%, and
6.2% for mild, moderate, and severe anaemia, respectively [64], in South Africa, prevalences
are mostly higher at 26.7%, 41.1%, and 4.3% [12]. In this study, 59.5% of those with moder-
ate/severe anaemia were PLWHIV. The majority had low mean CD4 counts, unsuppressed
VLs, and CDC Stage 3, which are indicative of advanced HIV disease and a high risk of
anaemia. The low prevalence of reported OIs other than TB in this study may be due to
limited investigations. Compared to low- and middle-income countries in Latin America and
Asia, adults in Sub-Saharan Africa have markedly lower incidences of Pneumocystis jirovecii
pneumonia and cerebral toxoplasmosis in ART-naïve patients [65], most likely due to a lack of
resources for diagnosis rather than a true difference in infection rates [66].
Patients with severe anaemia were three times more likely to have a diagnosis of TB
compared to the other major diagnostic categories (HIV, cancer, and CKD) as demonstrated
in the multivariate analysis (Table 6, which only considers patients with laboratory moder-
ate or severe anaemia). While Table 7 shows that in patients with TB and severe anaemia,
Hb was higher in those with TB compared to those without, the multivariate analysis
is the most accurate summation due to accounting for multiple variables. Table 7 is an
analysis of indices to assist in characterising findings in HIV and TB, which are the most
prevalent causes, compared to all other causes in patients with moderate/severe anaemia.
In PLWHIV with moderate (but not severe) anaemia, Hb was significantly lower compared
to those without HIV. HIV frequently causes impaired haematopoiesis, and anaemia is
the most common manifestation, increasing in frequency and severity with disease pro-
gression [63]. PLWHIV with moderate or severe anaemia had overall low CD4 counts
(median values < 200 cells/µL) indicative of more advanced disease [67]. PLWHIV with
TB and severe anaemia had lower CD4 counts and higher VL compared to those without
TB. This is consistent with HIV as the most potent immunosuppressive risk factor for
active TB [68]. HIV infection was associated with a slightly lower RPI that is not clinically
significant. Patients with or without HIV or TB and moderate and severe anaemia had
normal MCV and low MCHC (indicating hypochromia). However, MCHC is considered
of little clinical relevance in interpreting anaemias [69]. Median TSAT was also very low
(<16%) in both moderate and severe anaemia without HIV or TB, which is suggestive of
absolute ID [56]. Serum iron was lower in severe anaemia without HIV or TB. Serum iron
is depressed in both ID and AI and cannot differentiate between the two conditions [70].
Serum transferrin was significantly lower in patients with moderate and severe anaemia
and HIV or TB. It is an acute-phase reactant that deceases in inflammation but increases in
ID [41]. Ferritin and CRP, both inflammatory markers, were significantly higher in patients
with either HIV or TB and moderate or severe anaemia. CRP can be used for diagnosis of
pulmonary TB [71] and as an indicator of disease severity [72] and higher mortality [73].
Median CRP levels in patients with TB in this study, i.e., 146 mg/L (IQR 214 mg/L) in
moderate and 163 mg/L (IQR 122 mg/L) in severe anaemia, were considerably higher than
those of 40 mg/L (IQR 83 mg/L) in U.K. [72] and 17.3 (±37.2 mg/L, SD) in Taiwanese [73]
studies and indicative of more severe disease, possibly due to delayed diagnoses.
CKD stages 3–5 were present in 6.5% of patients with moderate and severe anaemia.
Prevalences of CKD stages 3–5 in Africa are 4.6% in the general population and 13% in
high-risk populations, e.g., HIV, diabetes, and hypertension [74], compared with 10.6% in
the general population worldwide [75]. The low prevalence of CKD may be due to the
Int. J. Environ. Res. Public Health 2023, 20, 3584 12 of 17

strict application of the criteria to define CKD, i.e., abnormalities of kidney structure or
function, present for >3 months [46], and non-performance of regular renal evaluations, in
patients at high risk, e.g., diabetes mellitus as elsewhere in South Africa [76], and the non-
availability of prior patient records with creatinine results. The actual prevalence is most
likely double this, as all creatinine-based GFR-estimating equations underestimate GFR
in African populations [77]. While cystatin C-based equations may be superior, cystatin
C is unaffordable in clinical practice in developing countries [78]. In this study, 32% with
CKD had IDA. This compares to a 35.3% IDA prevalence in non-dialysis CKD outpatients
in Johannesburg [79] and 15–72.8% in developed countries [80]. It is important that IDA is
recognised and treated to improve quality of life [81]. Most patients with stable CKD can
be managed in primary care [82].
In patients with cancer, one of six had IDA. The prevalences of anaemia and ID in
different cancers was reported as 29–46% and 7–42%, respectively, in developed countries
(ID prevalence at 60% was highest in colorectal cancer) [5]. No patient had colorectal cancer
in this study.
There was a low overall prevalence (2.5%) of vitamin B12 deficiency (<147.6 pmol/L)
compared to 8.8% in non-pregnant women of childbearing age elsewhere in South Africa [83].
While NHLS normal values for both sexes were 133–675 pmol/L, the lower limit of nor-
mal cannot be used as a cut-off to define deficiency due to frequent false-positive and
false-negative results [36] Methylmalonic acid and homocysteine levels are required to
confirm vitamin B12 deficiency [36], but testing was not mandated in the public-sector
guidelines [84]. As seen in this study, neuropsychiatric disorders due to deficiency often
occur without haematological changes [36]. While vitamin B12 deficiency can be found
in up to 30% of PLWHIV [85], there is little evidence of overt clinical disease [86,87]. A
minority of patients with vitamin B12 deficiency exhibited macrocytic changes. This is well
documented, particularly in patients with iron deficiency or inflammation [88]. Vitamin
B12 values were significantly higher in patients at MRH compared to the CHCs. Levels
were higher also in moderate and severe anaemia in PLWHIV and patients with TB. There
are reports of higher mortality with elevated vitamin B12 levels, namely >250 pmol/L [89]
and >700 pmol/L [90] by mechanisms yet to be elucidated.
Low community folate-deficiency prevalences of 0.2% [91] and zero in reproductive-
age women [83] were reported in South Africa following folate fortification in 2003. The
higher prevalence (6.2%) in this study may be due to inadequate nutrition during illness.
The majority (57.5%) of patients had multiple causes of anaemia compared with 13.6%
of elderly inpatients in South Africa [18] and 32.5% in Italy [52]. This relates to the high
levels of infectious diseases (TB and HIV) in this study population.
Approaches in the evaluation of anaemia are traditional evaluation using the MCV
and RPI [92,93] and biochemical tests [16,23,40]. South African national guidelines advise
categorising anaemia based on MCV and, if microcytic, testing for ID and, if macrocytic,
testing for vitamin b12 and folate deficiency [33,94]. As shown in this and other studies [95],
this approach will miss many patients with nutrient deficiencies and those with multiple
causes. With a substantial prevalence of ID, this study supports the recommendation
that iron status needs evaluation in every patient with anaemia [16,32] but also adds the
evaluation of folate and vitamin B12 deficiencies. In areas of high TB and HIV prevalence,
TB (pulmonary and extra-pulmonary) should be excluded as a cause of moderate and
severe anaemia [12,22]. Overall, a combination of MCV/RPI, biochemical, HIV, and TB
testing would seem the optimal approach for moderate and severe anaemia evaluation in
the study population.
The strengths of this study are that causes of anaemia were investigated compre-
hensively, using biochemical criteria and clinical assessment, and in the context of daily
practice in primary care and a district hospital setting. An updated pragmatic definition of
ID was used compared with previous studies. The cross-sectional study design is suited to
estimate the prevalence of anaemia in the study population [96].
Int. J. Environ. Res. Public Health 2023, 20, 3584 13 of 17

Limitations include missing data due to patient, research assistant, and laboratory
factors. Patients had multiple non-interoperable records, making it difficult to obtain
historical data. However, there was no indication of a systematic pattern that might
invalidate the analysis. Some patients were on drugs associated with anaemia. Due to the
cross-sectional study design (with absence of follow-up), it is not possible to state with
certainty that any drug was responsible for anaemia. TB was likely responsible for more
causes of anaemia, as patients with probable TB on clinical assessment were excluded
from analysis. In retrospect, our sample size could have been optimised by using the two-
proportions formula with a power of 0.8 instead of using the single-proportion formula,
which resulted in only 25.5% of the initial participants being eligible for the final analyses.
The findings are not generalisable but may be transferrable to similar settings.

5. Conclusions
In summary, HIV, ID, and TB were the most prevalent causes of moderate and severe
anaemia. Anaemia of inflammation was the most common type of anaemia. Patients with
severe anaemia were three times more likely to have TB. Changes in RBC indices do not
reliably predict nutritional deficiencies. Biochemical tests should routinely be performed to
assess iron, folate, and vitamin B12 deficiencies.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijerph20043584/s1.
Author Contributions: Conceptualization: P.Y. and D.O.; methodology, P.Y., D.O. and S.A.M.; vali-
dation, D.O. and S.A.M.; formal analysis, S.A.M.; investigation, M.M., J.I., R.K., K.A.F.N. and D.O.;
resources, B.O., E.B.-B., S.V. and K.A.F.N.; data curation, D.O. and S.A.M.; writing—original draft
preparation, D.O.; writing—review and editing, P.Y. and R.K.; visualization, S.A.M.; supervision,
P.Y.; project administration, P.Y.; funding acquisition, P.Y. All authors have read and agreed to the
published version of the manuscript.
Funding: The research was supported by an unrestricted grant from the Discovery Foundation (Ref
034609/2015), South Africa, awarded to P Yogeswaran.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and the protocol was approved by the Health Research Committee of the Faculty of
Health Sciences of Walter Sisulu University (035/2017). Permission to undertake the study was
obtained from the Department of Health (EC_2017RP30_423).
Informed Consent Statement: Written informed consent was obtained from all subjects before they
participated in the study.
Data Availability Statement: The data presented in this study are available in article or Supplemen-
tary Materials.
Acknowledgments: The authors acknowledge the cooperation of participants in this study and the
research assistants, Anita Hlatshaneni, Anele Lutango, Vuyokazi Mbonyana, and JN Xiva.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. WHO. The Global Prevalence of Anaemia in 2011; World Health Organization: Geneva, Switzerland, 2015.
2. Safiri, S.; Kolahi, A.-A.; Noori, M.; Nejadghaderi, S.A.; Karamzad, N.; Bragazzi, N.L.; Sullman, M.J.; Abdollahi, M.; Collins, G.S.;
Kaufman, J.S. Burden of anemia and its underlying causes in 204 countries and territories, 1990–2019: Results from the Global
Burden of Disease Study 2019. J. Hematol. Oncol. 2021, 14, 185. [CrossRef] [PubMed]
3. Fishbane, S.; Pollack, S.; Feldman, H.I.; Joffe, M.M. Iron indices in chronic kidney disease in the National Health and Nutritional
Examination Survey 1988–2004. Clin. J. Am. Soc. Nephrol. 2009, 4, 57–61. [CrossRef] [PubMed]
4. Anand, I.S.; Gupta, P. Anemia and iron deficiency in heart failure: Current concepts and emerging therapies. Circulation 2018, 138,
80–98. [CrossRef] [PubMed]
5. Aapro, M.; Österborg, A.; Gascón, P.; Ludwig, H.; Beguin, Y. Prevalence and management of cancer-related anaemia, iron
deficiency and the specific role of i.v. iron. Ann. Oncol. 2012, 23, 1954–1962. [CrossRef] [PubMed]
Int. J. Environ. Res. Public Health 2023, 20, 3584 14 of 17

6. Yates, J.; Logan, E.; Stewart, R. Iron deficiency anaemia in general practice: Clinical outcomes over three years and factors
influencing diagnostic investigations. Postgrad. Med. J. 2004, 80, 405–410.
7. McCartney, D.; Shine, B.; Hay, D.; Lasserson, D.S. The evaluation of anaemia in an older primary care population: Retrospective
population-based study. BJGP Open 2017, 1, bjgpopen17X101157. [CrossRef] [PubMed]
8. Boennelykke, A.; Jensen, H.; Falborg, A.Z.; Granfeldt Østgård, L.S.; Hansen, A.T.; Christensen, K.S.; Vedsted, P. Diagnostic
workup of cancer in patients with new-onset anaemia: A Danish cohort study in general practice. Scand. J. Prim. Health Care 2021,
39, 391–402. [CrossRef]
9. Shisana, O.; Labadarios, D.; Rehle, T.; Simbayi, L.; Zuma, K.; Dhansay, A.; Reddy, P.; Parker, W.; Hoosain, E.; Naidoo, P.; et al.
South African National Health and Nutrition Examination Survey (SANHANES-1); HSRC Press: Cape Town, South Africa, 2013.
10. National Department of Health (NDoH); Statistics South Africa (Stats SA); South African Medical Research Council (SAMRC);
ICF. South Africa Demographic and Health Survey 2016; National Department of Health: Pretoria, South Africa; Rockville, MD,
USA, 2019.
11. Stats, S. Mid-Year Population Estimates 2021; Statistical Release P0302 Statistics South Africa: Pretoria, South Africa, 2021.
12. Kerkhoff, A.D.; Wood, R.; Cobelens, F.G.; Gupta-Wright, A.; Bekker, L.-G.; Lawn, S.D. The predictive value of current haemoglobin
levels for incident tuberculosis and/or mortality during long-term antiretroviral therapy in South Africa: A cohort study. BMC
Med. 2015, 13, 70. [CrossRef]
13. SAMRC; HSRC; NICD. The First National TB Prevalence Survey South Africa 2018; Short Report; South african Medical Research
Council: Cape Town, South Africa, 2020.
14. Gelaw, Y.; Getaneh, Z.; Melku, M. Anemia as a risk factor for tuberculosis: A systematic review and meta-analysis. Environ. Health
Prev. Med. 2021, 26, 13. [CrossRef]
15. Barzegari, S.; Afshari, M.; Movahednia, M.; Moosazadeh, M. Prevalence of anemia among patients with tuberculosis: A systematic
review and meta-analysis. Indian J. Tuberc. 2019, 66, 299–307.
16. Weiss, G.; Ganz, T.; Goodnough, L.T. Anemia of inflammation. Blood J. Am. Soc. Hematol. 2019, 133, 40–50. [CrossRef] [PubMed]
17. Walsh, C.M.; Hattingh, Z.; Veldman, F.J.; Bester, C.J. Iron status and anaemia of chronic disease in HIV-infected African women in
Mangaung, Bloemfontein. S. Afr. Fam. Pract. 2010, 52, 55–59. [CrossRef]
18. Van Staden, A.M.; Weich, D.J. Retrospective analysis of the prevalence and causes of anaemia in hospitalised elderly patients. S.
Afr. Fam. Pract. 2015, 57, 297–299. [CrossRef]
19. Claster, S. Biology of anemia, differential diagnosis, and treatment options in human immunodeficiency virus infection. J. Infect.
Dis. 2002, 185 (Suppl. 2), S105–S109. [CrossRef] [PubMed]
20. Lewis, D.K.; Whitty, C.J.M.; Walsh, A.L.; Epino, H.; Broek, N.R.; Letsky, E.A.; Munthali, C.; Mukiibi, J.M.; Boeree, M.J. Treat-
able factors associated with severe anaemia in adults admitted to medical wards in Blantyre, Malawi, an area of high HIV
seroprevalence. Trans. R. Soc. Trop. Med. Hyg. 2005, 99, 561–567. [CrossRef] [PubMed]
21. Kerkhoff, A.D.; Lawn, S.D.; Schutz, C.; Burton, R.; Boulle, A.; Cobelens, F.J.; Meintjes, G. Anemia, Blood Transfusion Requirements
and Mortality Risk in Human Immunodeficiency Virus-Infected Adults Requiring Acute Medical Admission to Hospital in South
Africa. Open Forum Infect. Dis. 2015, 2, ofv173. [CrossRef] [PubMed]
22. Mntonintshi, M.; O’Mahony, D.; Mabunda, S.; Namugeyi, K.A.F. Undiagnosed tuberculosis in patients with HIV infection who
present with severe anaemia at a district hospital. Afr. J. Prm. Health Care Fam. Med. 2017, 9, a1406.
23. Kerkhoff, A.D.; Meintjes, G.; Opie, J.; Vogt, M.; Jhilmeet, N.; Wood, R.; Lawn, S.D. Anaemia in patients with HIV-associated TB:
Relative contributions of anaemia of chronic disease and iron deficiency. Int. J. Tuberc. Lung Dis. 2016, 20, 193–201. (In English)
[CrossRef]
24. Stats, S.A. Poverty Mapping in South Africa. Applying Small Area Estimation Techniques Using IES 2010/11 and Census 2011; Statistics
South Africa: Pretoria, South Africa, 2018.
25. Joubert, G.; Ehrlich, R.; Katzenellenbogen, J.M.; Karim, S.A. Epidemiology: A Research Manual for South Africa, 2nd ed.; Oxford
University Press Southern Africa: Cape Town, South Africa, 2007.
26. Whitley, E.; Ball, J. Statistics review 4: Sample size calculations. Crit Care 2002, 6, 335–341. [CrossRef]
27. WHO. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. In Vitamin and Mineral Nutrition
Information System; WHO/NMH/NHD/MNM/11.1; World Health Organization: Geneva, Switzerland, 2011.
28. NDoH. Implementation of the Universal Test and Treat Strategy for HIV Positive Patients and Differentiated Care for Stable Patients;
Circular dated 16 August 2016; National Department of Health: Pretoria, South Africa, 2016.
29. NDoH. Essential Drugs Programme. In Primary Healthcare Standard Treatment Guidelines and Essential Medicines List, 5th ed.;
National Department of Health, Republic of South Africa: Pretoria, South Africa, 2014.
30. NDoH. National Consolidated Guidelines for the Prevention of Mother-to-Child Transmission of HIV (PMTCT) and the Management of
HIV in Children, Adolescents and Adults; Department of Health: Pretoria, South Africa, 2015.
31. Heller, T.; Wallrauch, C.; Goblirsch, S.; Brunetti, E. Focused assessment with sonography for HIV-associated tuberculosis (FASH):
A short protocol and a pictorial review. Crit. Ultrasound J. 2012, 4, 21. [CrossRef]
32. Adamson, J.W.; Longo, D.L. Anemia and Polycythemia. Chapter 77. In Harrison’s Principles of Internal Medicine, 19e; Kasper, D.,
Fauci, A., Hauser, S., Longo, D., Jameson, J., Loscalzo, J., Eds.; McGraw-Hill: New York, NY, USA, 2015.
33. NDoH. Essential Drugs Programme. In Hospital Level (Adults) Standard Treatment Guidelines and Essential Medicines List, 5th ed.;
Department of Health: Pretoria, South Africa, 2019.
Int. J. Environ. Res. Public Health 2023, 20, 3584 15 of 17

34. NICE. Anaemia Management in People with Chronic Kidney Disease; National Institute for Health and Care Excellence: London,
UK, 2015.
35. Brugnara, C. Diagnostic Approaches to Iron Deficient States and Iron Deficiency Anemia. Haema 2019, 10, 2–14.
36. Stabler, S.P. Clinical practice. Vitamin B12 deficiency. N. Engl. J. Med. 2013, 368, 149–160. [CrossRef]
37. WHO. Serum and Red Blood Cell Folate Concentrations for Assessing Folate Status in Populations; WHO/NMH/NHD/EPG/15.01;
World Health Organization: Geneva, Switzerland, 2015.
38. Mast, A.E.; Blinder, M.A.; Gronowski, A.M.; Chumley, C.; Scott, M.G. Clinical utility of the soluble transferrin receptor and
comparison with serum ferritin in several populations. Clin. Chem. 1998, 44, 45–51. [CrossRef]
39. Camaschella, C.; Girelli, D. The changing landscape of iron deficiency. Mol. Asp. Med. 2020, 75, 100861. [CrossRef]
40. Cappellini, M.D.; Musallam, K.M.; Taher, A.T. Iron deficiency anaemia revisited. J. Intern. Med. 2020, 287, 153–170. [CrossRef]
41. Ganz, T. Anemia of Inflammation. N. Engl. J. Med. 2019, 381, 1148–1157. [CrossRef]
42. Ponikowski, P.; Voors, A.A.; Anker, S.D.; Bueno, H.; Cleland, J.G.; Coats, A.J.; Falk, V.; González-Juanatey, J.R.; Harjola, V.-P.;
Jankowska, E.A. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur. Heart J. 2016, 37,
2129–2200. [CrossRef] [PubMed]
43. Aapro, M.; Beguin, Y.; Bokemeyer, C.; Dicato, M.; Gascón, P.; Glaspy, J.; Hofmann, A.; Link, H.; Littlewood, T.; Ludwig, H.
Management of anaemia and iron deficiency in patients with cancer: ESMO Clinical Practice Guidelines. Ann. Oncol. 2018, 29
(Suppl. 4), iv96–iv110. [CrossRef] [PubMed]
44. NICE. Chronic Kidney Disease: Assessment and Management (NG203); National Institute for Health and Care Excellence: London,
UK, 2021.
45. KDIGO. KDIGO clinical practice guidelines for acute kidney injury. Kidney Int. Suppl. 2012, 2, 1–138.
46. KDIGO CKD Work Group. KDIGO 2012. Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney
Disease. Kidney Int. 2013, 3, S1–S150. [CrossRef]
47. Mikhail, A.; Brown, C.; Williams, J.A.; Mathrani, V.; Shrivastava, R.; Evans, J.; Isaac, H.; Bhandari, S. Clinical Practice Guideline:
Anaemia of Chronic Kidney Disease; The Renal Association: Bristol, UK, 2017.
48. Selik, R.M.; Mokotoff, E.D.; Branson, B.M.; Owen, M.; Whitmore, S.; Hall, H.I. Revised surveillance case definition for HIV
infection—United States, 2014. Morb. Mortal. Wkly. Rep. Recomm. Rep. 2014, 63, 1–10.
49. Mash, B.; Fairall, L.; Adejayan, O.; Ikpefan, O.; Kumari, J.; Mathee, S.; Okun, R.; Yogolelo, W. A morbidity survey of South African
primary care. PLoS ONE 2012, 7, e32358. [CrossRef]
50. Payne, C.F.; Davies, J.I.; Gomez-Olive, F.X.; Hands, K.J.; Kahn, K.; Kobayashi, L.C.; Tipping, B.; Tollman, S.M.; Wade, A.; Witham,
M.D. Cross-sectional relationship between haemoglobin concentration and measures of physical and cognitive function in an
older rural South African population. J. Epidemiol. Community Health 2018, 72, 796–802. [CrossRef] [PubMed]
51. Whitehead, R.D., Jr.; Mei, Z.; Mapango, C.; Jefferds, M.E.D. Methods and analyzers for hemoglobin measurement in clinical
laboratories and field settings. Ann. N. Y. Acad. Sci. 2019, 1450, 147–171. (In English) [CrossRef]
52. Randi, M.L.; Bertozzi, I.; Santarossa, C.; Cosi, E.; Lucente, F.; Bogoni, G.; Biagetti, G.; Fabris, F. Prevalence and Causes of Anemia
in Hospitalized Patients: Impact on Diseases Outcome. J. Clin. Med. 2020, 9, 950. [CrossRef] [PubMed]
53. Guralnik, J.M.; Eisenstaedt, R.S.; Ferrucci, L.; Klein, H.G.; Woodman, R.C. Prevalence of anemia in persons 65 years and older in
the United States: Evidence for a high rate of unexplained anemia. Blood 2004, 104, 2263–2268. [CrossRef]
54. Guyatt, G.H.; Oxman, A.D.; Ali, M.; Willan, A.; McIlroy, W.; Patterson, C. Laboratory diagnosis of iron-deficiency anemia. J. Gen.
Intern. Med. 1992, 7, 145–153. [CrossRef]
55. Garcia-Casal, M.N.; Pasricha, S.-R.; Martinez, R.X.; Lopez-Perez, L.; Pena-Rosas, J.P. Are current serum and plasma ferritin
cut-offs for iron deficiency and overload accurate and reflecting iron status? A systematic review. Arch. Med. Res. 2018, 49,
405–417. [CrossRef]
56. Cacoub, P.; Vandewalle, C.; Peoc’h, K. Using transferrin saturation as a diagnostic criterion for iron deficiency: A systematic
review. Crit. Rev. Clin. Lab. Sci. 2019, 56, 526–532. [CrossRef]
57. Girelli, D.; Marchi, G.; Camaschella, C. Anemia in the elderly. HemaSphere 2018, 2, e40. [CrossRef]
58. Mentzer, W. Differentiation of iron deficiency from thalassaemia trait. Lancet 1973, 301, 882. [CrossRef] [PubMed]
59. Bird, A.; Ellis, P.; Wood, K.; Mathew, C.; Karabus, C. Inherited haemoglobin variants in a South African population. J. Med. Genet.
1987, 24, 215–219. [CrossRef] [PubMed]
60. Loonat, S.B.; Naran, N.H.; Thein, S.L.; Alli, N.A. Alpha-thalassaemia trait as a cause of unexplained microcytosis in a South
African population. S. Afr. Med. J. 2016, 106, 276–279. [CrossRef] [PubMed]
61. Krause, A.; Wainstein, T.; Essop, F.B.; Goodyear, Q. Testing for haemoglobinopathies in Johannesburg, South Africa: A 30-year
review. S. Afr. Med. J. 2013, 103, 989–993. [CrossRef]
62. Wu, D.-W.; Li, Y.-M.; Wang, F. How long can we store blood samples: A systematic review and meta-analysis. EBioMedicine 2017,
24, 277–285. [CrossRef]
63. Durandt, C.; Potgieter, J.C.; Khoosal, R.; Nel, J.G.; Herd, C.L.; Mellet, J.; Rossouw, T.; Pepper, M.S. HIV and haematopoiesis. S. Afr.
Med. J. 2019, 109 (Suppl. 1), S41–S46. [CrossRef]
64. Cao, G.; Wang, Y.; Wu, Y.; Jing, W.; Liu, J.; Liu, M. Prevalence of anemia among people living with HIV: A systematic review and
meta-analysis. eClinicalMedicine 2022, 44, 101283. [CrossRef]
Int. J. Environ. Res. Public Health 2023, 20, 3584 16 of 17

65. Low, A.; Gavriilidis, G.; Larke, N.; B-Lajoie, M.R.; Drouin, O.; Stover, J.; Muhe, L.; Easterbrook, P. Incidence of Opportunistic
Infections and the Impact of Antiretroviral Therapy Among HIV-Infected Adults in Low- and Middle-Income Countries: A
Systematic Review and Meta-analysis. Clin. Infect. Dis. 2016, 62, 1595–1603. [CrossRef]
66. Morris, A.; Lundgren, J.D.; Masur, H.; Walzer, P.D.; Hanson, D.L.; Frederick, T.; Huang, L.; Beard, C.B.; Kaplan, J.E. Current
epidemiology of Pneumocystis pneumonia. Emerg. Infect. Dis. 2014, 10, 1713. [CrossRef]
67. Ingle, S.M.; May, M.T.; Gill, M.J.; Mugavero, M.J.; Lewden, C.; Abgrall, S.; Fätkenheuer, G.; Reiss, P.; Saag, M.S.; Manzardo, C.;
et al. Impact of Risk Factors for Specific Causes of Death in the First and Subsequent Years of Antiretroviral Therapy Among
HIV-Infected Patients. Clin. Infect. Dis. 2014, 59, 287–297. [CrossRef]
68. Narasimhan, P.; Wood, J.; MacIntyre, C.R.; Mathai, D. Risk factors for tuberculosis. Pulm. Med. 2013, 2013, 828939. [CrossRef]
69. Merritt, B.Y. Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC); Medscape, WebMD
LLC: New York, NY, USA, 2019; Available online: https://emedicine.medscape.com/article/2054497 (accessed on 4 October 2022).
70. Camaschella, C. Iron-Deficiency Anemia. N. Engl. J. Med. 2015, 372, 1832–1843. [CrossRef] [PubMed]
71. Yoon, C.; Chaisson, L.H.; Patel, S.M.; Allen, I.E.; Drain, P.K.; Wilson, D.; Cattamanchi, A. Diagnostic accuracy of C-reactive protein
for active pulmonary tuberculosis: A meta-analysis. Int. J. Tuberc. Lung Dis 2017, 21, 1013–1019. (In English) [CrossRef] [PubMed]
72. Brown, J.; Clark, K.; Smith, C.; Hopwood, J.; Lynard, O.; Toolan, M.; Creer, D.; Barker, J.; Breen, R.; Brown, T.; et al. Variation in
C—Reactive protein response according to host and mycobacterial characteristics in active tuberculosis. BMC Infect. Dis. 2016,
16, 265. [CrossRef]
73. Huang, C.-T.; Lee, L.-N.; Ho, C.-C.; Shu, C.-C.; Ruan, S.-Y.; Tsai, Y.-J.; Wang, J.-Y.; Yu, C.-J. High serum levels of procalcitonin and
soluble TREM-1 correlated with poor prognosis in pulmonary tuberculosis. J. Infect. 2014, 68, 440–447. [CrossRef] [PubMed]
74. Kaze, A.D.; Ilori, T.; Jaar, B.G.; Echouffo-Tcheugui, J.B. Burden of chronic kidney disease on the African continent: A systematic
review and meta-analysis. BMC Nephrol. 2018, 19, 125. [CrossRef]
75. Hill, N.R.; Fatoba, S.T.; Oke, J.L.; Hirst, J.A.; O’Callaghan, C.A.; Lasserson, D.S.; Hobbs, F.R. Global prevalence of chronic kidney
disease–a systematic review and meta-analysis. PLoS ONE 2016, 11, e0158765. [CrossRef]
76. Webb, E.M.; Rheeder, P.; Van Zyl, D.G. Diabetes care and complications in primary care in the Tshwane district of South Africa.
Prim. Care Diabetes 2015, 9, 147–154. [CrossRef]
77. Fabian, J.; Kalyesubula, R.; Mkandawire, J.; Hansen, C.H.; Nitsch, D.; Musenge, E.; Nakanga, W.P.; Prynn, J.E.; Dreyer, G.; Snyman,
T. Measurement of kidney function in Malawi, South Africa, and Uganda: A multicentre cohort study. Lancet Glob. Health 2022,
10, e1159–e1169. [CrossRef]
78. Niang, A.; Luyckx, V.A. Measuring kidney function: The voice of Africa. Lancet Glob. Health 2022, 10, e1080–e1081. [CrossRef]
79. Nalado, A.M.; Mahlangu, J.N.; Waziri, B.; Duarte, R.; Paget, G.; Olorunfemi, G.; Naicker, S. Ethnic prevalence of anemia and
predictors of anemia among chronic kidney disease patients at a tertiary hospital in Johannesburg, South Africa. Int. J. Nephrol.
Renov. Dis. 2019, 12, 19–32. (In English) [CrossRef] [PubMed]
80. Babitt, J.L.; Eisenga, M.F.; Haase, V.H.; Kshirsagar, A.V.; Levin, A.; Locatelli, F.; Małyszko, J.; Swinkels, D.W.; Tarng, D.-C.; Cheung,
M.; et al. Controversies in optimal anemia management: Conclusions from a Kidney Disease: Improving Global Outcomes
(KDIGO) Conference. Kidney Int. 2021, 99, 1280–1295. [CrossRef]
81. KDIGO. Kidney Disease: Improving Global Outcomes (KDIGO) Anaemia Work Group clinical practice guideline for anemia in
chronic kidney disease. Kidney Int. Suppl. 2012, 2, 279–335.
82. John, R.; Webb, M.; Young, A.; Stevens, P.E. Unreferred chronic kidney disease: A longitudinal study. Am. J. Kidney Dis. 2004, 43,
825–835. [CrossRef]
83. Modjadji, S.; Alberts, M.; Mamabolo, R. Folate and iron status of South African non-pregnant rural women of childbearing age,
before and after fortification of foods. S. Afr. J. Clin. Nutr. 2007, 20, 89–93. [CrossRef]
84. NDOH. Standard Treatment Guidelines and Essential Medicines List for South Africa. Hospital Level, Adults, 4th ed.; NDOH: Pretoria,
South Africa, 2015.
85. Vishnu, P.; Aboulafia, D.M. Haematological manifestations of human immune deficiency virus infection. Br. J. Haematol. 2015,
171, 695–709. (In English) [CrossRef] [PubMed]
86. Paltiel, O.; Falutz, J.; Veilleux, M.; Rosenblatt, D.S.; Gordon, K. Clinical correlates of subnormal vitamin B12 levels in patients
infected with the human immunodeficiency virus. Am. J. Hematol. 1995, 49, 318–322. [CrossRef] [PubMed]
87. Hepburn, M.J.; Dyal, K.; Runser, L.A.; Barfield, R.L.; Hepburn, L.M.; Fraser, S.L. Low serum vitamin B12 levels in an outpatient
HIV-infected population. Int. J. STD AIDS 2004, 15, 127–133. [CrossRef]
88. Metz, J. Haematological implications of folate food fortification. S. Afr. Med. J. 2013, 103 (Suppl. 1), 978–981. [CrossRef]
89. Flores-Guerrero, J.L.; Minović, I.; Groothof, D.; Gruppen, E.G.; Riphagen, I.J.; Kootstra-Ros, J.; Kobold, A.M.; Hak, E.; Navis, G.;
Gansevoort, R.T. Association of plasma concentration of vitamin B12 with all-cause mortality in the general population in the
Netherlands. JAMA Netw. Open 2020, 3, e1919274. [CrossRef]
90. Wolffenbuttel, B.H.R.; Heiner-Fokkema, M.R.; Green, R.; Gans, R.O.B. Relationship between serum B12 concentrations and
mortality: Experience in NHANES. BMC Med. 2020, 18, 307. [CrossRef] [PubMed]
91. Labadarios, D. National Food Consumption SurveyFortification Baseline (NFCS-FB): South Africa, 2005; Department of Health: Pretoria,
South Africa, 2007.
92. Greer, J.P.; Arber, D.A.; Glader, B.; List, A.F.; Means, R.T.J.; Paraskevas, F.; Rodgers, G.M. Wintrobe’s Clinical Hematology, 13th ed.;
Lippincott, Williams & Wilkins: Philadelphia, PA, USA, 2014.
Int. J. Environ. Res. Public Health 2023, 20, 3584 17 of 17

93. Kasper, D.; Fauci, A.; Hauser, S.; Longo, D.; Jameson, J.; Loscalzo, J. Harrison’s Principles of Internal Medicine, 19e; McGraw-Hill:
New York, NY, USA, 2015.
94. NDoH. Essential Drugs Programme. In Primary Healthcare Standard Treatment Guideline and Essential Medicine List, 7th ed.; National
Department of Health: Pretoria, South Africa, 2020.
95. Petrosyan, I.; Blaison, G.; Andrès, E.; Federici, L. Anaemia in the elderly: An aetiologic profile of a prospective cohort of 95
hospitalised patients. Eur. J. Intern. Med. 2012, 23, 524–528. [CrossRef] [PubMed]
96. Sedgwick, P. Cross sectional studies: Advantages and disadvantages. BMJ 2014, 348, g2276. [CrossRef]

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