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Hookworm Infection among School Age Children in Kintampo North
Municipality, Ghana: Nutritional Risk Factors and Response to Albendazole
Treatment
Article  in  The American journal of tropical medicine and hygiene · July 2013
DOI: 10.4269/ajtmh.12-0605 · Source: PubMed
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Am. J. Trop. Med. Hyg., 89(3), 2013, pp. 540–548
doi:10.4269/ajtmh.12-0605
Copyright © 2013 by The American Society of Tropical Medicine and Hygiene
Hookworm Infection among School Age Children in Kintampo North Municipality, Ghana:
Nutritional Risk Factors and Response to Albendazole Treatment
Debbie Humphries,* Benjamin T. Simms, Dylan Davey, Joseph Otchere, Josephine Quagraine, Shawn Terryah,
Samuel Newton, Elyssa Berg, Lisa M. Harrison, Daniel Boakye, Michael Wilson, and Michael Cappello
Program in International Child Health, Department of Pediatrics and School of Public Health, Yale School of Medicine,
New Haven, Connecticut; Department of Parasitology, Noguchi Memorial Institute for Medical Research,
University of Ghana, Accra, Ghana
Abstract. Children (n = 812) 6–11 years of age attending 16 schools in the KintampoNorthMunicipality of Ghana were
screened for participation in a study on hookworm infection, nutrition, and response to albendazole. The prevalence
of Necator americanus hookworm infection (n = 286) was 39.1%, and significant predictors of infection included age,
malaria parasitemia, lack of health care, school area, levels of antibodies against hookworm, and low consumption
of animal foods. The cure rate after a single dose (400 mg) albendazole was 43%, and the mean fecal egg count reduction
rate was 87.3%. Data for an in vitro egg hatch assay showed a trend toward reduced albendazole susceptibility in post-
treatment hookworm isolates (P = 0.06). In summary, hookworm infection is prevalent among school age children in the
Kintampo North Municipality and animal food intake inversely correlates with infection status. Modest cure rates and fecal
egg count reduction rates reinforce the need for further investigation of potential benzimidazole resistance in Ghana.
INTRODUCTION observed a cure rate of 61% and an individual arithmetic mean
fecal egg count reduction (FECR) rate of 82%, both of which
More than half a billion persons worldwide, including 156mil- were lower than anticipated.
lion children, are infected with blood-feeding hookworms.1–3 We report results of a follow up study conducted in KNM
Hookworm infection increases risk of anemia and causes nega- in 2010. The purpose of this study was to validate treatment
tive effects on growth, iron status, and cognition in children results from our previous study and test the hypothesis that
with high intensity infections.4–6 Hookworm is most prevalent nutrition predicts infection status and response to single-dose
in resource poor areas,7 where limited access to sanitation, albendazole. The field study also provided an opportunity
sewage treatment, and use of night soil in agriculture contribute to implement in vitro testing of hookworm isolates for suscep-
to transmission.8–10 Although rarely fatal, the morbidity associ- tibility to albendazole, thereby providing baseline data to track
ated with chronic infection may increase susceptibility to other emerging resistance in KNM.
tropical diseases, including HIV, tuberculosis, and malaria.11–15
Mass drug administration (MDA) of anthelminthics is a cor-
nerstone of the World Health Organization (WHO) strategy METHODS
for integrated control of seven neglected tropical diseases.16
With regard to hookworm, long-term follow-up from semi- Ethical approval. This study was approved by the Yale Uni-
annual treatment programs of pre-school age and school age versity Human Investigations Committee and the Institutional
children shows a decrease in intensity of infection (³ 80% Review Boards at the Noguchi Memorial Institute for Medical
in Zanzibar), but smaller effects on prevalence.2,17–19 Similar Research, the Ghana Health Service, and the Scientific Review
results from early studies suggested that MDA decreases inten- Committee and the Institutional Ethics Committee at the
sity and prevalence of infection over time,2 although a recent Kintampo Health Research Center. In addition, District Minis-
Cochrane review concluded that there is limited evidence try of Health representatives and District Ministry of Education
of consistent effects of MDA on nutrition and cognition among representatives approved the study and assisted in communica-
school children.20 This recent suggestion that the benefits tionwith participating schools.
of school based deworming may be overstated has generated Participant enrollment. In June 2010, 16 schools located
significant dialogue regarding the potential for MDA to control along a 90-km stretch of the major highway north of
geohelminth infections globally. Kintampo were invited to participate in a research study.
In 2007, a community-based study in the Kintampo North Each school provided a list of all students 6–11 years of age.
Municipality (KNM) of Ghana identified a hookworm preva- Duplicate height and weight measurements were obtained for
lence of 45% (n = 292), most notable for the absence of other each child by using a stadiometer and electronic scale. Height
geohelminths.21 Adults with hookworm had a significantly for age (HAZ), weight for age (WAZ), and BMI for age
lower average body mass index (BMI), despite the predomi- (BAZ) z-scores were calculated for each participant by using
nance of low intensity infection (< 1,000 eggs per gram [epg] the WHO anthropometric calculator (Anthro Plus version
of feces). In children, we noted a hookworm prevalence of 56%, 1.0.3, http://who-anthroplus.software.informer.com/1.0/). Stu-
andmanywere co-infectedwithPlasmodiumfalciparum.Among dents with HAZ £ −1.80 or HAZ ³ −0.10 were invited to par-
all persons treated with single-dose albendazole (400 mg), we ticipate in the study. One child was randomly selected from
each household to eliminate the potential for clustering effects
at the household level. HAZ cutoffs for inclusion were derived
from the screening sample to obtain a final sample size of
* 300 persons (with an extra 10% included at the initial selectionAddress correspondence to Debbie Humphries, Yale School of Pub-
lic Health, 60 College Street, PO Box 208034, New Haven, CT 06520. to enable nonparticipation) from the 812 screened. In the
E-mail: debbie.humphries@yale.edu selected group of children, approximately half had low HAZ
540
HOOKWORM INFECTION AMONG SCHOOL AGE CHILDREN IN GHANA 541
scores and half had high or normal HAZ scores. This sampling single dose of albendazole treatment (400 mg) (Wormzap; GR
method was designed to investigate the relationship between Industries Limited, Accra, Ghana) under direct observation.
chronic undernutrition (stunting) and prevalence of hookworm Ten to fourteen days after treatment, fecal samples were col-
infection within a geographic region by using a sample size lected for two consecutive days and analyzed as described
commensurate with the resources of the study team. After above. The FECR rates were calculated as individual and
receipt of permission from community leaders, teachers were group arithmetic means by using the methods recommended
asked to invite the parents of the potential participants for a by Vercruysse and others.24
meeting to explain the study and ask for consent. The final Egg hatch assay: analysis of in vitro anthelminthic activity
study population consisted of 286 participants from 16 commu- of albendazole. The susceptibility of human hookworm iso-
nities (Figure 1). lates to albendazole was assessed before and after treatment
Sample collection and processing. Fecal collection con- using an in vitro EHA.25,26 Stock solutions of albendazole
tainers and directions for use were distributed to individual (Sigma, St. Louis, MO) were prepared in methanol (5 mg/
students, and collected the following day. After receipt of the mL) and further diluted in distilled water. Approximately 50
first fecal sample, students were asked to provide a second eggs isolated from individual study participants (n = 71) were
fecal sample the following day. Microscopy was used to iden- suspended in a final volume of 100 mL and added to indi-
tify and count parasite ova by using the Kato-Katz method vidual wells of a 96 well microtiter plate containing 100 mL
(Vestergaard-Frandsen, Lausanne, Switzerland) as outlined of albendazole solution (final concentrations = 0, 0.1, 1, 2,
by WHO.22 Hookworm eggs were purified from positive fecal and 5 mg/mL). Plates were incubated at ambient tempera-
samples by serial suspension and centrifugation in 0.9% NaCl, ture, and after 48 hours the number of hatched first-stage
0.015% Brij-35, and 2.18 M NaNO3 (specific gravity = 1.185), hookworm larvae was counted using light microscopy. When
followed by filtration through an 80-mm filter, using the post-treatment fecal egg counts showed a sufficient numbers
method reported by Reiss and colleagues.23 Egg concentra- of eggs, susceptibility was also evaluated from samples col-
tions were adjusted to 50 eggs/100 mL for use in the egg hatch lected after albendazole treatment (n = 14). Samples in which
assay (EHA; see below); remaining eggs were frozen for the positive control wells (no albendazole) showed a hatch
DNA analysis. Each hookworm infected child was referred for rate < 75% rate were excluded from analysis.
Speciation of hookworm isolates using polymerase chain
reaction. Genomic DNA was extracted from frozen hook-
worm eggs collected from 103 persons using the QIAamp
DNA stool kit (QIAGEN, Carlsbad, CA). A nested polymer-
ase chain reaction (PCR) amplification method27 was used
to amplify species-specific sequences within the internal tran-
scribed spacer of ribosomal DNA. Reaction products were
subjected to agarose gel electrophoresis, and successful ampli-
fication was indicated by an 870-base pair product for Necator
americanus and a 690-base pair product for Ancylostoma
duodenale. The PCR products were excised from the gel and
the DNA extracted by using QIAGEN gel extraction kit
reagents and protocols (QIAGEN). The DNA sequence was
confirmed in a subset of PCR products.
Blood and serum analysis. Approximately 1 mL of blood
was aliquoted for automated complete blood count analysis
conducted at the Kintampo Health Research Center. A malaria
rapid diagnostic test kit (First Response Malaria Ag HRP-2;
Premier Medical Corporation Ltd., Watchung, NJ) was used
to test for P. falciparum antigens. Among children who had a
positive result for the rapid diagnostic test, thick and thin blood
smears were prepared to verify the presence of Plasmodium
spp. by using light microscopy according to WHO recommen-
dations.22 All study participants were asymptomatic for malaria
at the time of screening. Antigen-specific IgG responses against
A. ceylanicum adult worm excretory–secretory (ES) proteins
were measured using a described enzyme-linked immuno-
sorbent assay (ELISA).21,28 Levels of serum reactivity (optical
density at 405 nm) for the study population were categorized
by quartile for statistical analysis. One community chose not
to provide blood samples and was excluded from analysis.
Household questionnaire.The survey instrument was adapted
from the Demographic and Health Surveys, the Household
Dietary Diversity Score for Measurement of Household Food
Access: Indicator Guide, and the Escala Latinoamericana y
Figure 1. Participation selection, recruitment and data collection, Caribena de Seguridad Alimentaria Household Food Insecurity
29–31
Ghana. HAZ = height for age. scale. The survey instrument was translated into the local
542 HUMPHRIES AND OTHERS
language (Twi) and back-translated into English by native sis (Mann-Whitney U test, Kruskal Wallis test) was used when
speakers to confirm accuracy. Questions included birth date, comparing non-normally distributed variables, including base-
household socioeconomic characteristics, parental education line epg, mean cell volume, and red cell distribution width.
and occupation, water and sanitation access, access to health
care and vaccinations, history of anthelminthic treatment, bed
net use, food insecurity, hunger, and dietary diversity. Inter- RESULTS
viewers from the Rural Health Training School in Kintampo
administered the surveys in the local language. A list of house- Intestinal parasite assessment of study population. Fecal
hold assets was used to construct an absolute wealth index32 samples were obtained from 279 persons, representing a range
based on the number of assets, including a tile floor, use of 7–36 students/school. The overall prevalence of hookworm
of advanced cooking fuel (not charcoal, straw or wood), elec- in the study population was 39.1% (109/279), and of those that
tricity, radio, TV, phone, refrigerator, bike, car or motorcycle, were infected, 82.6% (90/109) were classified as having light
land, cow, horse or donkey, goat or sheep, pig, poultry, bank or infections (< 1,000 epg).36Other intestinal parasites were present
savings account, improved water source, improved toilet). The in 15% of the population, includingHymenolepis nana (10.7%),
wealth index was tested in the models as a continuous variable Taenia spp., and other unidentified tapeworm species. Ascaris
and a categorical variable (tertiles of wealth). lumbricoides and Trichuris trichiura were not detected. The
Determination of anthropometric status. Child birth dates prevalence of hookworm infection varied significantly across
were confirmed with a household member as part of the house- the schools, ranging from 15% to 70%.
hold survey. In cases of discordance between dates provided by Socioeconomic status and risk of hookworm infection.
households and schools, analyses were based on the household Fewer than half of the households used an improved water
birth date when available, and the school birth date if no source (well, rainwater, borehole, spring) or latrine (Table 1).
household birth date was known. A categorical age variable Ownership of mosquito nets was high (78%). Most of the male
was used to minimize the effect of potential misclassification. and female household heads (80%) and female caregivers
An examination of the effects of age onHAZ,WAZ, and BAZ (63%) were farmers. More than two-thirds of the female care-
showed that BAZ was less affected by age variation in children givers (69.7%) and heads of household (67.3%) reported
6–11 years of age, and thus was the most robust measure no schooling. Among the socioeconomic variables considered,
of nutritional status given the known error in the age data. parental occupation (farmer), larger household size, and own-
Therefore, HAZ and WAZ were not included in the analysis. ership of a pig were associated with significantly higher preva-
Dietary diversity, household food insecurity, and consump- lence of hookworm infection in bivariate analyses (Table 1).
tion of animal source foods. Children and caregivers were Children who had not recalled seeing a health care provider
asked about consumption of foods the previous day and the within the past year had significantly higher prevalence of hook-
previous week. Eleven nutrient-rich food groups (excluding con- worm infection (Table 1), and this effect remained significant
diments and sugar) were transformed into a binary dietary in multivariate analysis (Table 2).
diversity variable, with participants either above or below the Anthropometric and nutritional indicators. Only 3.5% (10)
group mean. Consumption of animal source foods (ASF) were of the participants had BAZ scores < −2.0. Almost all (³ 95%)
extracted into a subscale, and transformed into a binary ASF of the children reported consuming grain, vegetables, roots/
variable with participants above or below the group mean. tubers, and fish on at least a weekly basis, and ³ 85% reported
Household food insecurity was categorized according to stan- having consumed those food groups the previous day. Seventy-
dard methods for the Latin American and Caribbean Food nine percent of the households reported some food insecurity,
Insecurity Scale,33 which has been used in Ghana.34 A hunger and>30%reportedmoderate-to-severefoodinsecurity(Table3).
subscale was extracted from the Escala Latinoamericana y Animal source foods (meat, organ meat, eggs, dairy, and fish)
Caribena de Seguridad Alimentaria questions, which included showed significant variation with household food security and
five questions pertaining to reductions in food quantity. wealth (P < 0.05). The average blood hemoglobin level among
Statistical analysis. All data generated from this study were study participants was 10.5 g/dL, and 71.8% of the children were
stored in Microsoft (Redmond, WA) Excel (2007), and statisti- below theWHOdesignation for anemia (11.5 g/dL).37Compared
cal analysis was conducted with SPSS (Chicago, IL) versions with children with blood hemoglobin levels ³ 11.5g/dL, those
17.0 and 19.0. An initial review of descriptive statistics preceded with levels < 11.5 g/dL had significantly lower mean cell volumes
bivariate analysis of factors to determine their relationship to (80.5 versus 83.3; P < 0.01) and significantly higher mean RDWs
baseline hookworm infection and response to single-dose (14.0 versus 13.5; P < 0.05), which is consistent with microcytic
albendazole. Initial variables tested were derived from a theo- or iron deficiency anemia.
retical framework of factors supported by the literature to affect Nutritional status, dietary intake, and risk of hookworm
exposure and susceptibility to hookworm infection. Variables infection. No anthropometric measures were associated with
representing key constructs from the theoretical framework hookworm infection at baseline, including BAZ (Table 3). How-
were analyzed to identify a subset for analysis that minimized ever, lower food insecurity and above average consumption
the effects of collinearity. Salient variables from the univariate of ASF were each associated with a reduced odds of hookworm
analysis were used in a multivariate analysis adjusting for clus- infection (P < 0.01 for both). The relationship between ASF
tering within schools35 by applying logistic regression analyses consumption and hookworm infection remained significant
within generalized estimating equation algorithms. In addition in multivariate analysis, with children in the below average ASF
to controlling for the school effect, schools were grouped into group having a three-fold higher risk for hookworm infection
four geographic clusters of four schools each that were used at baseline (P < 0.001) (Table 2).
in the analysis. Final models included control variables and vari- Malaria infection. Using the malaria rapid diagnostic test
ables of statistical significance (P < 0.05). Nonparametric analy- kit, we found that 84.7% (210/249) of participants were positive
HOOKWORM INFECTION AMONG SCHOOL AGE CHILDREN IN GHANA 543
Table 1
Socioeconomic and health indicators of study population for hookworm infection, Ghana*
Characteristic All Hookworm negative (n = 170) Hookworm positive (n = 109) P†
Age < 8 years 32.4 (91) 37.6 (64) 24.3 (27) 0.056
Age 8–9 years 34.2 (96) 32.9 (56) 36.9 (41)
Age ³ 10 years 33.1 (93) 29.4 (50) 38.7 (43)
Female 51.6 (145) 55.3 (94) 45.9 (51) 0.125
Improved water sources
Drinking 45.9 (129) 47.6 (81) 43.2 (48) 0.469
Cooking 43.4 (122) 44.7 (76) 41.4 (46) 0.589
Bathing 43.4 (122) 45.3 (77) 40.5 (45) 0.432
Use of a latrine 34.9 (98) 35.9 (61) 33.3 (37) 0.661
Mosquito net ownership 77.2 (217) 75.3 (128) 80.2 (89) 0.340
Bicycle 85.1 (239) 82.9 (141) 88.3 (98) 0.219
Car/motorcycle 19.6 (55) 18.8 (32) 20.7 (23) 0.695
Pig 12.1 (34) 6.5 (11) 20.7 (23) < 0.001
Bank account 44.5 (125) 42.9 (73) 46.8 (52) 0.520
Household head occupation
Farmer 80.4 (226) 75.7 (128) 88.3 (98) 0.023
Small trader/other 14.2 (40) 17.2 (29) 9.9 (11)
None 5.0 (14) 7.1 (12) 1.8 (2)
Household head schooling
No schooling 67.3 (189) 63.0 (104) 76.6 (85) 0.076
Primary school 11.4 (32) 13.3 (22) 9.0 (10)
More than primary school 29.5 (55) 23.6 (39) 14.4 (16)
Female caregiver occupation
Farmer 63.2 (175) 53.9 (89) 80.4 (86) < 0.001
Small trader/other 26.7 (74) 35.8 (59) 14.0 (15)
None 8.3 (23) 10.3 (16) 5.6 (6)
Female caregiver schooling
No schooling 79.4 (193) 77.1 (111) 82.4 (84) 0.105
Primary school 14.0 (34) 13.2 (19) 15.7 (16)
More than primary school 7.6 (16) 9.7 (14) 2.0 (2)
No. children £ 5 years of age 25.6 (72) 32.4 (55) 15.3 (17) < 0.001
1 child £ 5 years of age 32.4 (91) 34.1 (58) 29.7 (33)
³ 2 children £ 5 years of age 42.0 (118) 33.5 (57) 55.0 (61)
Above average wealth 49.5 (139) 48.2 (82) 51.7 (57) 0.558
Household size 8.1 ± 4.1 7.51 ± 3.8 8.95 ± 4.5 0.004
Mosquito nets/person 0.39 ± 0.20 0.41 ± 0.2 0.37 ± 0.2 0.174
Deworming in the last year 15.4 (42) 19.3 (32) 9.3 (10) 0.026
Health care access within last year 50.0 (132) 70.5 (93) 29.5 (39) 0.002
More than one year 33.3 (88) 54.5 (48) 45.5 (40)
Never 16.7 (44) 43.2 (19) 56.8 (25)
*Cells indicate the proportion of children that are hookworm negative (or positive) for each characteristic. Values are values are % (no.) for categorical variables and mean ± SD for
continuous variables.
†By chi-square test or t test.
and all were asymptomatic. The malaria species was defini- tion between antibodies to A. ceylanicum ES and hookworm
tively identified as P. falciparum on 97.5% of the smears. infection status.21
Based on univariate analysis, children with malaria were more Response to treatment with albendazole. One hundred
likely to have hookworm infection (P = 0.026), and the preva- nine children who were positive for hookworm infection at
lence of co-infection in the study population was 35.1%. baseline were treated with a single 400-mg oral dose of
In multivariate analysis, malaria infection remained signifi- albendazole and 107 (96.4%) provided follow-up fecal sam-
cantly associated with hookworm infection, with the odds ratio ples. As shown in Table 4, the albendazole cure rate was
for hookworm infection increasing with higher malaria parasite 43% (46 of 107), and all children had < 1,000 epg at follow-
density (Table 2). up. The FECR rate was 87.3%, based on the arithmetic
Hookworm antibody levels. The level of serum IgG against mean of group egg counts before and after treatment, and
A. ceylanicum excretory secretory antigens was also positively 70.3% using individual arithmetic means. There was a
associated with hookworm infection status (P < 0.001, by uni- slightly higher baseline epg in persons who remained posi-
variate analysis). As shown in Figure 2, in each of the four tive post-treatment (P = 0.045) based on a nonparametric
regions surveyed, the level of antigen-specific IgG, as mea- rank test. However, analysis based on a theoretical frame-
sured by serum ELISA, was lower among those who were work of predictors of response to albendazole did not iden-
negative for hookworm at baseline compared with those who tify any significant predictors of response to treatment
were infected. Based on multivariate analysis (Table 2), those among the variables collected. The BMI for age score was
with antibody levels in the second, third. Or fourth highest not associated with response to treatment, nor were house-
quartile were 2.22 (P = 0.25), 6.47 (P = 0.001), or 11.76 (P < hold food security, hunger, dietary diversity, or pre-treat-
0.001) times more likely to be infected at baseline compared ment anti-hookworm antibody levels.
with those in the lowest quartile of antibody responses. This In vitro susceptibility of hookworm isolates. An in vitro
result is consistent with our prior investigation of the correla- EHA was used to characterize the susceptibility of hookworm
544 HUMPHRIES AND OTHERS
Table 2
Risk factors for hookworm infection, Ghana*
Adjusted 95% confidence
Characteristic odds ratio† interval P
Age £ 7 years (reference)
Age 8–9 years 1.54 0.75–3.20 0.245
Age ³ 10 years 1.45 0.80–2.64 0.224
Wealth (reference = lowest tertile)
Highest tertile 0.98 0.31–3.08 0.973
Middle tertile 0.85 0.42–1.70 0.645
Female 0.65 0.30–1.40 0.272
Health care (reference = within 1 yr)
> 1 year 1.50 0.69–3.28 0.306
Never 2.52 1.32–4.80 < 0.01
School Region South (reference)
Mid South 6.60 2.90–14.98 < 0.001
Mid North 5.96 2.92–2.16 < 0.001
North 7.24 1.64–31.92 < 0.01
Malaria (reference = 0 parasites/mL)
1–499 parasites/mL 1.26 0.69–3.28 0.539
500–1,999 parasites/mL 2.25 1.14–4.45 0.02
³ 2,000 parasites/mL 5.50 2.10–14.41 < 0.01
Anti-ES IgG (reference = lowest quartile)
Second lowest quartile 2.22 1.10–4.48 0.25
Third quartile 6.47 2.11–19.87 0.001
Highest quartile 11.76 3.32–41.67 < 0.001
Below average animal source foods 3.23 1.83–5.71 < 0.001
*For adjusted model, n = 212. ES = excretory–secretory.
†Adjusted for all other variables in this model and for clustering within schools. Figure 2. IgG concentration by hookworm status and school
region, Ghana. Median IgG levels (horizontal bar) are presented as a
isolates to albendazole.38 As shown in Figure 3, pre-treatment function of infection status (positive versus negative) across the four
school regions. Bottom of the box represents the 25th percentile, and
samples (n = 71) exhibited reduced overall hatching rates in the top represents the 75th percentile. Error bar represents the 5–95%
presence of increasing concentrations of albendazole, ranging percentile, and solid circles are outlier data points. OD = optical density.
from a mean ± SD hatch rate of 92.1 ± 8.0% at 0.1 mg/mL to
23.2 ± 17.2% at 5 mg/mL. At the highest concentration of drug
(5 mg/mL), the median hatch rate for post-treatment samples samples), suggesting that exposure to albendazole may select for
was 2.3 fold higher in post-treatment samples than in those parasites with reduced susceptibility to albendazole.
collected before treatment (34.0% versus 14.8% (Figure 3). Speciation of hookworm isolates using PCR. To define the
The differences in hatch rate were not statistically significant predominant hookworm species causing infection in KNM,
(P = 0.06), presumably because of the small number of post- genomic DNA was extracted from hookworm eggs isolated
treatment samples (n= 12).Within the groupofmatched samples from persons who were positive by fecal microscopy. Coding
(n= 10), weobservedan increase (³ 10%) inhatchrateat5mg/mL sequences corresponding to the internal transcribed spacer
of albendazole in three samples, a decrease in three samples, and 2 region of ribosomal DNA were amplified by using a nested
no substantial change in four samples. However, a similar dispar- PCR protocol.27 Definitive speciation results were obtained
ity in egg hatch rates was also noted at 2 mg/mL of albendazole from 94 (91.2%) of 103 samples that were tested by PCR. All
(73.6% in post-treatment samples versus 50.6% in pre-treatment 94PCRresults identifiedN.americanus, and twopersons (1.9%)
Table 3
Anthropometry and nutritional indicators of study population for hookworm infection, Ghana*
Hookworm negative Hookworm positive
Characteristic All, % (no.) (n = 170), % (no.) (n = 109), % (no.) P
BAZ mean (SD) −0.652 (0.77) −0.71 (0.72) −0.57 (0.83) 0.13
BAZ > 0 19.2 (54) 31 (18.2) 20.7 (23) 0.794
BAZ −0.99–0 48.8 (137) 48.8 (83) 48.6 (54)
BAZ −1.99–1.0 28.5 (80) 30.0 (51) 26.1 (29)
BAZ £ −2.0 3.5 (10) 2.9 (5) 4.5 (5)
Hemoglobin, mean (SD) 10.5 (1.20) 10.55 (1.22) 10.47 (1.16) 0.609
RDW, mean (SD) 13.88 (1.27) 13.83 (1.27) 13.97 (1.28) 0.401
MCV, mean (SD) 81.01 (6.01) 81.41 (5.60) 80.36 (6.60) 0.203
Household food secure 21.2 (52) 19.2 (28) 24.2 (24) 0.009
Some food insecurity 46.1 (113) 54.1 (79) 34.3 (34)
Moderate food insecurity 16.7 (41) 15.8 (23) 18.2 (18)
Severe food insecurity 15.9 (39) 11.0 (16) 23.2 (23)
No household hunger 58.4 (164) 61.2 (104) 54.1 (60) 0.029
Some hunger 28.5 (80) 30.0 (51) 26.1 (29)
Severe hunger 13.2 (37) 8.8 (15) 19.8 (22)
Above average dietary diversity 39.6 (84) 44.5 (53) 33.3 (31) 0.098
Above average ASF groups 42.5 (107) 51.6 (79) 28.3 (28) < 0.001
*BAZ = body mass index for age; RDW = red cell distribution width; MCV= mean cell volume; ASF = animal source foods.
HOOKWORM INFECTION AMONG SCHOOL AGE CHILDREN IN GHANA 545
Table 4 Ultimately, control of hookworm infection will likely require
Treatment response to albendazole (400 mg) for hookworm a multi-faceted approach that includes efforts to identify intrin-
infection, Ghana sic or modifiable host and parasite factors that mediate suscep-
Characteristic Value tibility to infection (and reinfection), as well as the response
Baseline prevalence 39.1% (109/279) to therapy. Nutritional factors, such as dietary content, may
Cure rate 43.0% (46/107) influence risk of infection, and supplementary feeding has been
Arithmetic mean egg count* proposed as a potential method to control gastrointestinal
Baseline 503.2 ± 625.6
Post-treatment 63.0 ± 133.2 nematodes in livestock.
42 This study confirms that low-level
Fecal egg count reduction rate (%) consumption of animal source foods groups is significantly
Group arithmetic mean (n = 107) 87.3 associated with human hookworm infection status, along with
Individual arithmetic mean (n = 107) 70.3 ± 64.1 older age, higher malaria parasite density, lack of access
*Arithmetic mean egg count values are mean ± SD eggs per gram (of feces). to health care, school region, and higher hookworm antibody
levels, even after controlling for wealth and sex. To our knowl-
were found to have a mixed infection with N. americanus and edge, increased prevalence of hookworm infection in school
A.duodenale.No isolatedA.duodenale infectionsweredetected. children who consume below average amounts of animal source
food groups has not been reported.
Animal source foods are an important source of dietary
DISCUSSION protein, as well as micronutrients such as iron, vitamin A,
Mass drug administration is recommended byWHO for con- vitamin B-12, riboflavin, calcium, and zinc.
43 Low consump-
trol of four geohelminth infections.16 The goal of preventive tion of animal source foods has been linked to deficiencies
chemotherapy using MDA is to reduce the intensity of infec- in multiple micronutrients, particularly in children from low-
44
tion (i.e., worm burden) among persons, which also has the income countries. Numerous cross-sectional studies have
potential to reduce transmission of infection within disease- confirmed an association between poor nutritional status and
45–48
endemic communities. Concerns about reduced effectiveness intestinal helminth infections, and recent studies suggest
of commonly used anthelminthics have focused on the poten- that malnutrition increases susceptibility to helminth infection
49–51
tial emergence of hookworm resistance, especially against the and re-infection. Our data are also supported by animal
benzimidazoles mebendazole and albendazole.39–41 studies showing that protein supplementation confers protection
against nematode infections.52–54 Thus, the finding in KNM
of lower rates of hookworm among children who consume
higher amounts of animal source foods provides further evidence
that nutritional status may affect susceptibility to hookworm
infection in this population. Importantly, the data analyses do
not enable assignment of causality, but nonetheless point to an
interesting association between dietary intake and hookworm
infection status.
As in a prior study,21 children in KNM who were positive
for malaria had a higher prevalence of hookworm infection at
baseline, and the risk of hookworm increased with the level
of malaria parasitemia. The high prevalence of malaria and
helminth co-infections among school children is consistent
with prior analyses of geographic distribution and age-related
patterns of the two infections.55,56 Recent reviews suggest that
helminth infections have specific effects on the epidemiology
and pathogenesis of malaria.57 Overall, however, reports
of the effects of helminth co-infection on malaria prevalence
and clinical phenotype vary. Cross-sectional studies of school
children found a higher prevalence of helminth infections
among children with P. falciparum58; although a study of young
children (6–23 months of age) found a lower prevalence of hel-
minth infections among children with malaria.59 A deworming
trial in Nigeria found a significantly lower rate of increase
in malaria prevalence and intensity among children who were
treated with albendazole.60 In Thailand, Ascaris co-infection
decreased the risk of cerebral malaria,61 although a prospective
community-based observational study by the same authors
Figure 3. Susceptibilityof hookworm isolates to albendazole before
and after treatment, Ghana. Top panel shows box plot of in vitro sus- found a higher risk of symptomatic malaria among helminth
ceptibility testing of hookworm isolates collected prior to deworming. infected individuals.
62 Other studies have found no association
Horizontal bars represent median hatch rate, error bars represent the between hookworm and malaria,63,64 demonstrating that inter-
5–95th percentile, and solid circles are outlier data points. Bottom pretation of results is confounded bymarked differences in study
panel shows individual subject egg hatch rates (solid circles) in the
m populations, presence of multiple helminths with varying inten-presence of 5 g/mL of albendazole for samples taken pre-treatment
and post-treatment. Horizontal bars represent median hatch rates for sities, and in the study definition of malaria used.
65 In general,
each data set. current opinion seems to hold that hookworm, in contrast
546 HUMPHRIES AND OTHERS
to other helminths (Ascaris, schistosomes), may increase malaria based in vitro analysis of albendazole susceptibility by using
susceptibility and clinical severity, although the issue has hookworm eggs harvested from individual persons before and
not been settled.57 Given that there are > 400 million cases after treatment. The data are notable for two findings. First,
of P. falciparum malaria each year,66 and that > 500 million and consistent with the diversity in cure rates and FECR
persons are infected with hookworm,2 a better understanding rates, we noted a wide range of susceptibility among isolates
of the dynamics of co-infection could lead to the development between study participants, which suggests that the populations
of more effective control measures for both diseases. of hookworm within KNM are distinct in terms of benzimid-
In this study, we confirmed previous observations21 that anti- azole susceptibility. To date, few studies have applied in vitro
bodies directed at adult ES proteins from a laboratory strain susceptibility testing to human hookworm isolates by using the
of A. ceylanicum correlate with infection status. This finding EHA. In 1997, de Clercq and others
73 reported a lower than
further validates A. ceylanicum as a useful tool for characteriz- expected response to mebendazole in Mali, as well as EHA
ing human hookworm responses because those with the highest data suggesting that the human isolates were less susceptible
antibody responses were > 11 times more likely to be infected than a laboratory strain of N. americanus. Subsequently, Kotze
25
at the time of sampling, a difference that was highly statistically and others successfully defined 50% lethal dose values
significant (P < 0.001). Although the hookworm ES ELISA for thiabendazole and albendazole by using a small number
lacks specificity to predict infection status on an individual of (pre-treatment) N. americanus isolates from Papua New
26
basis (Figure 2), this assay could potentially serve as an effec- Guinea. Albonico and others used an in vitro assay similar
tive screening tool to monitor control programs at the com- to the one reported here to characterize susceptibility of human
munity level. If deworming or other intervention programs isolates from Pemba Island to mebendazole and thiabendazole.
Important differences with our study include the fact that they
resulted in a substantial reduction in prevalence, we would
pooled 10 participant samples before testing, and that suscepti-
anticipate that seroreactivity would also decrease over time.
bility to albendazole was not tested.
To define the molecular epidemiology of hookworm in KNM,
Despite the limited number of matched samples in our study,
we undertook studies to identify the species of hookworm caus-
the trend toward reduced albendazole susceptibility observed
ing infection in this region. Using a PCR-based assay, we deter-
in samples collected post-treatment (Figure 3) suggests that
mined that N. americanus and A. duodenale are endemic to
treatment selects for isolates that are more resistant to the
KNM, although infection with N. americanus is significantly
drug. The data also demonstrate the utility of field-based
more prevalent (100% of infected persons versus 2%). The pre-
in vitro susceptibility testing, and show that samples from indi-
dominance of N. americanus in Ghana is consistent with prior
67–69 vidual persons can be broadly distinguished based on drugstudies conducted in northern Ghana. To our knowledge,
susceptibility. Work is currently underway aimed at defining
however, this is the first report of the species distribution of molecular markers that correlate with albendazole susceptibil-
hookworm in the more centrally located KNM. These data can ity with treatment response and molecular markers.
now serve as a baseline for future studies of the molecular epide- In summary, hookworm infection is prevalent among school
miology of hookworm in central Ghana. age children in KNM, and dietary intake of animal source
Current recommendations of WHO call for single-dose ther- foods inversely correlates with infection status. Although no
apy with one of four anthelminthics, including albendazole, as specific host factors were associated with treatment failure,
part of preventive chemotherapy for soil-transmitted nema- we observed that post-treatment isolates of N. americanus
todes. Reported cure rates for hookworm infection from single exhibited reduced in vitro susceptibility to albendazole.
dose (400 mg) therapy with albendazole range from 40% to Because of the modest cure rate and fecal egg count reduction
100%, and an average cure rate of 78.4%.70 In light of the rate observed, further investigation of potential benzimid-
prevailing opinion that intensity, i.e., worm burden, is the most azole resistance in Ghana is warranted.
significant factor mediating the clinical sequelae of hookworm
infection, it has been proposed that cure rate is not as impor- Received September 28, 2012. Accepted for publication May 1, 2013.
tant a measure of deworming efficacy as the FECR rate, which
Published online July 8, 2013.
more closely correlates with the impact of treatment on worm
burden.24,71 In the present study, we observed an albendazole Acknowledgments: We thank the people of Kintampo North Munic-
ipality for their participation in the study, and the Noguchi Memorial
cure rate of 43%, which is less than the cure rate of 61% we
Institute for Medical Research and the Ghana–Yale Partnership for
reported in a study of children and adults in Kintampo.21 The Global Health for support.
FECR rate ranged from 70.3%, when calculated by using indi-
Financial support: This study was supported by National Institutes
vidual arithmetic means, to 87.3% by using group arithmetic of Health grant RO1AI099623 (Michael Wilson), a Wilbur Downs
means of epg (Table 4). These FECR rates are similar to what International Health Fellowship from the Yale School of Public
has been reported from other disease-endemic populations.72 Health (BenjaminT. Simms), aBenjaminH.KeanTravelingFellowship
A recent collaborative report, which included representation in Tropical Medicine from the American Society of Tropical Medicine
and Hygiene (Dylan Davey), and a Medical Scholars Program Award
from WHO, suggested that hookworm FECR rates > 90%
from the InfectiousDiseases Society ofAmerica (DylanDavey).
(measured by using group arithmetic means) after albendazole
treatment should increase concern about the possibility of resis- Authors’ addresses: Debbie Humphries, Yale School of Public Health,
24 New Haven, CT, E-mail: debbie.humphries@yale.edu. Benjamin T.tance, a position with which we concur. By validating our Simms and Shawn Terryah, Department of Epidemiology of Microbial
initial observations from 2007, we have now established that Disease, Yale School of Public Health, New Haven, CT, E-mails:
the cure rate and FECR rate in Kintampo have reached a level simmsb@gmail.com and shawn.terryah@gmail.com. Dylan Davey,
requiring careful monitoring of deworming effectiveness. Josephine Quagraine, Elyssa Berg, Lisa M. Harrison, and Michael
Cappello, Department of Pediatrics, Yale School of Medicine, New
Having been alerted to the potential emergence of benz-
Haven, CT, E-mails: dylan.davey@yale.edu, josephine.quagraine@yale
imidazole resistance by results from the prior study,21 we .edu, elyssa.berg@yale.edu, lisa.harrison@yale.edu, and michael
integrated into the 2010 field study application of a field- .cappello@yale.edu. Joseph Otchere, Daniel Boakye, and Michael
HOOKWORM INFECTION AMONG SCHOOL AGE CHILDREN IN GHANA 547
Wilson, Department of Parasitology, Noguchi Memorial Institute for helminthiases in Zanzibar in the context of national helminth
Medical Research, Accra, Ghana, E-mails: jotchere@noguchi.mimcom control programs.Am J TropMedHyg 81: 1071–1078.
.org, dboakye@noguchi.mimcom.org and mwilson@noguchi.mimcom 18. De Rochars MB, Direny AN, Roberts JM, Addiss DG, Radday J,
.net. Samuel Newton, Department of Epidemiology, Kintampo Health Beach MJ, Streit TG, Dardith D, Lafontant JG, Lammie PJ,
Research Centre, Kintampo, Ghana, E-mail: samkofinewton@ 2004. Community-wide reduction in prevalence and intensity
yahoo.com. of intestinal helminths as a collateral benefit of lymphatic fila-
riasis elimination programs. Am J Trop Med Hyg 71: 466–470.
19. Phommasack B, Saklokham K, Chanthavisouk C, Nakhonesid-
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