Clinical features, risk factors, and impact of antibiotic treatment of diarrhea caused by Shigella in children less than 5 years in Manhiça District, rural Mozambique.

OBJECTIVES: During the period from December 2007 to November 2012, the epidemiology of diarrhea caused by Shigella was studied among children <5 years of age residing in Manhiça District, Southern Mozambique.
MATERIALS AND METHODS: Children from 0 to 5 years with moderate-to-severe diarrhea (MSD) and less severe diarrhea (LSD) were enrolled along with matched controls (by age, gender, and neighborhood). Age-stratified logistic regression analyses were conducted to identify clinical features and risk factors associated with Shigella positivity in cases of diarrhea. The impact of antibiotic treatment was assessed for patients with known outcome.
RESULTS: A total of 916 cases of MSD and 1979 matched controls, and 431 cases of LSD with equal number of controls were enrolled. Shigella was identified as significant pathogen in both cases of MSD and LSD compared to their respective controls. Shigella was detected in 3.9% (17/431) of LSD compared to 0.5% (2/431) in controls (P=0.001) and in 6.1% (56/916) of MSD cases compared to 0.2% (4/1979) in controls (P<0.0001), with an attributable fraction of 8.55% (95% CI: 7.86-9.24) among children aged 12-23 months. Clinical symptoms associated to Shigella among MSD cases included dysentery, fever, and rectal prolapse. Water availability, giving stored water to child, washing hands before preparing baby's food, and mother as caretaker were the protective factors against acquiring diarrhea caused by Shigella. Antibiotic treatment on admission was associated with a positive children outcome.
CONCLUSION: Shigella remains a common pathogen associated with childhood diarrhea in Mozambique, with dysentery being a significant clinical feature of shigellosis. Adherence to the basic hygiene rules and the use of antibiotic treatment could contribute to the prevention of most of diarrhea due to Shigella.

Introduction

Diarrhea is the leading cause of mortality in children <5 years with its long-term impact on growth and cognitive development.1 Although the burden is greater in low-income populations, acute infectious diarrhea is also a common cause of outpatient visits and hospital admissions in high-income regions and is an important health problem globally.2 Associated risk factors for infection include poor living standards, overcrowding, inadequate sanitation, and poor hand hygiene. These factors may result in a significant disease burden and economic effect due to direct medical costs, loss of work, lower quality of life, and mortality.3

The high burden of the disease is also associated with the wide range of recognized enteric pathogens such as virus, bacteria, and parasites that may cause diarrhea.4 Among them, Shigella infections remain a global public health concern, in resource-limited countries where the disease may cause as many as 163 million episodes of diarrhea and over a million deaths annually with the majority (60%) occurring in children under 5.5 Due to the high disease burden and endemicity of Shigella in resource-limited countries, the WHO has set the development of candidate vaccines against Shigella as priority.6 Thus, considering the efficacy of Shigella vaccine, it is necessary to have reliable estimates of the burden and epidemiology of disease in targeted endemic areas, including age-specific incidence data and risk factors associated with Shigella infections.

Primarily, shigellosis is transmitted from person to person through the fecal–oral route, but it may also spread indirectly by fecal contamination of water or food.7 Signs/symptoms of individuals who are infected with Shigella expand fever, painful bloody diarrhea, and stomach cramps.8 WHO recommends that all episodes of diarrhea with blood in the stool must be treated with antibiotics.9 Antimicrobial therapies reduce the period and intensity of disease symptoms, decrease excretion of bacteria, and prevent potentially lethal complications. Decreasing the bacterial load excreted by a child with dysentery also reduces the probability of fecal–oral transmission to close contacts, such as neighbors, friends, or members of the child’s household.10

In Mozambique, epidemiologic data on diarrhea caused by Shigella remain scarce and existing data are limited to few studies.11,12 Moreover, the contribution of Shigella to less severe diarrhea (LSD), the risk factors, and the impact of antibiotic treatment on patient outcome remain unknown. We hereby report the epidemiology of diarrhea due to Shigella among children <5 years of age enrolled between 2007 and 2012 in Manhiça district as part of the Global Enteric Multicenter Study.13

Materials and methods

Study area and population

The study was conducted by the Manhiça Health Research Centre (Centro de Investigação em Saúde de Manhiça – CISM) in Manhiça district, a rural area of Maputo province in southern Mozambique. The climate is subtropical with warm and rainy season from October to April and a fresh and dry season for the rest of the year. A round-the-clock morbidity surveillance system, covering pediatric inpatient and outpatient visits, was established in 1998 in the Manhiça District Hospital in a joint collaboration with CISM. Clinical data are routinely collected from all children under 15 years seeking health care. HIV infection is among the highest worldwide, with prevalence rates in women in childbearing age as high as 40% in the district.14 Diarrhea is the third leading cause of hospital admission among children aged 0–14 years and the fourth cause of death in children from 12 to 59 months.15 Additionally, since 1996, CISM has been running a Health Demographic Surveillance System (HDSS) for vital events and migrations in the population living within the study area covering approximately 95,000 inhabitants during the study period. In 2014, the study area was expanded to the whole district, and currently, 183,000 inhabitants are under DSS follow-up. Each person living within the DSS study area is issued a unique Permanent Identification Number that describes the geographic location, household number, and personal number within the household. A full description of the geographic and sociodemographic characteristics of the study community has been detailed elsewhere.16

Study design

The present analysis is part of a prospective case–control study of diarrhea conducted in Manhiça district from December 2007 to November 2012 to estimate the burden and etiology of moderate-to-severe diarrhea (MSD) in children <5 years of age to guide future interventions. To assess the role of Shigella in LSD, cases of LSD were enrolled from November 3rd 2011 to November 2nd 2012. Age in months was stratified in three strata (0–11, 12–23, and 24–59). Clinical and epidemiologic data were collected over the study period. Cases of diarrhea included in the study corresponded to children <5 years of age living within the HDSS area presenting to Manhiça district hospital with symptoms of diarrhea, defined as three or more loose stools in the last 24 hours. Clinicians assessed each child with diarrhea for eligibility; to be included, the episode had to be new (onset after ≥7 diarrhea-free days), acute (onset within the previous 7 days), and fulfill at least one of the following criteria for MSD: sunken eyes (confirmed by parent or caretaker as more than normal); loss of skin turgor (abdominal skin pinch with slow [≤2 seconds] or very slow [≥2 seconds] recoil); intravenous hydration administered or prescribed; dysentery (visible blood in loose stools); or admission to hospital with diarrhea or dysentery.17 For each child with diarrhea, one to three healthy control children (no story of diarrhea in the previous 7 days, matched by age, sex, and neighborhood) were randomly selected using the HDSS database and enrolled within 14 days of presentation of the corresponding index case.

Sample collection and laboratory analysis

Fecal samples from cases were collected within 12 hours of registration of the diarrheal episode, and control samples within 14 days after case enrolment. Once collected, samples were kept in a cool box until processed. Each fecal specimen comprised a whole stool specimen (in screw top fecal specimen cups carried in Styrofoam boxes with cold packs), a fecal swab in Modified Cary Blair medium in a plastic screw top test tube, and a fecal swab in buffered glycerol saline in a screw top test tube.13 Additionally, if antibiotics were to be given to patients before the production of stool sample, two rectal swabs were obtained for bacterial culture pending passage of the whole stool for the remaining assays. Stool samples were tested for the presence of bacterial, viral, and parasitic pathogens by culture, ELISA, and PCR as appropriate.18 In the case of Shigella, species were identified by colonies morphology upon culturing in MacConkey and XLD followed by slide agglutination with specific antisera (Denka Seiken Co., Ltd., Tokyo, Japan).

Data analysis

The software for analysis was Stata/SE version 14.1 (STATA Corporation, College Station, TX, USA) and the package coxphf from R version 3.2.2. All analyses were stratified by age. The frequency of Shigella isolation was calculated dividing the number of Shigella positivity by the total number of children enrolled over the study period with known culture result for Shigella. The pathogenicity of Shigella was assessed comparing the isolation rate between cases of diarrhea and control group. Logistic regression models were used to evaluate associations. All models were estimated with the penalized likelihood according to the Firth’s approach.19,20 Multivariable models were estimated by forward-stepwise selection from covariates with a P-value<0.20 in the crude models and no more than 5% of missing values. Significant levels for removal and addition in the multivariable models were 0.10 and 0.05, respectively, by Wald test. The association between MSD and Shigella was assessed at crude level for all pathogens and for Shigella adjusted for other significant pathogens and pairwise interactions between each one of them with Shigella. Analysis of sign/symptoms and sociodemographic factors associated with Shigella-MSD vs other types of MSD was performed among MSD cases. Interactions between significant covariates in the multivariable model were also assessed.

Ethical approval

The study is part of the Global Enteric Multi-Center Study (GEMS), which was approved by the Institutional Review Board at the University of Maryland School of Medicine, USA and the National Bioethics Committee for Health of Mozambique and Hospital Clinic, University of Barcelona, Spain. A written informed consent was obtained from children’s mothers or caretakers for participation in the study.

Results

Shigella and association with diarrhea

During the study period, a total of 916 cases and 1979 controls for MSD (December 9, 2007 to November 2, 2012), and 431 LSD cases and their respective 431 matched controls (November 3, 2011 to November 2, 2012) were enrolled. Shigella was isolated in 6.1% (56/916) of MSD cases compared to 0.2% (4/1979) in controls (P<0.0001) and in 3.9% (17/431) of LSD compared to 0.5% (2/431) in control group (Table 1). In addition, Shigella infection was predominantly detected during rainy season (October–March) compared to dry season (Figure 1). The incidence rate of Shigella-associated MSD was 0.61 per 100 child-year (95% CI: 0.48–0.74) with an attributable fraction of 8.55 (95% CI: 7.86–9.24) in children aged 12–23 months. A strong association of Shigella and MSD was found by both crude and pathogen-adjusted multivariate analysis for all age groups especially in children aged 24–59 months where adjusted odds ratio (aOR) of 190.92 (95% CI: 23.80–24565.84; P<0.0001) was observed as shown in Table 2.

Table 1

Detection rate of Shigella among children 0–59 months of age with diarrhea (MSD and LSD) and their matched controls in Manhiça district, December 2007 to November 2012

Age groups	Case/control		P-value
	Controls, n (%)	Cases, n (%)
MSD
0–11 months	1/1047 (0.1)	6/495 (1.2)	0.002
12–23 months	2/629 (0.3)	21/276 (7.6)	<0.0001
24–59 months	1/303(0.3)	29/145 (20)	<0.0001
Total	4/1979 (0.2)	56/916 (6.1)	<0.0001
LSD
0–11 months	0/155 (0)	1/155 (0.7)	0.317
12–23 months	1/175 (0.6)	10/175 (5.7)	0.006
24–59 months	1/101 (1)	6/101 (5.9)	0.054
Total	2/431 (0.5)	17/431 (3.9)	0.001

Abbreviations: LSD, less severe diarrhea; MSD, moderate-to-severe diarrhea.

Figure 1

Seasonal distribution of Shigella infection in children aged 0–59 months in Manhiça District (December 2007–November 2012).

Table 2

Crude and pathogen-adjusted multivariate analysis of the association of Shigella spp. with moderate-to-severe diarrhea among children under 5 years (December 2007–November 2012)

Variables	Group		Crude analysis		Adjusted analysis
	Controls, n (%)	Cases, n (%)	OR (95% CI)	P-value	Adjusted OR (95% CI)	P-value
0–11 months
Shigella spp.	1/1047 (0)	6/495 (1)	22.01 (2.44–2912.20)	0.0028	42 (93.96–5717.57)	0.0008
Cryptosporidium	96/1046 (9)	98/495 (20)	2.89 (2.07–4.06)	<0.0001	3.71 (2.54–5.50)	<0.0001
Giardia	177/1046 (17)	45/495 (9)	0.48 (0.33–0.68)	<0.0001	0.47 (0.31–0.69)	0.0001
Rotavirus	160/1046 (15)	217/495 (44)	5.91 (4.42–8.01)	<0.0001	6.37 (4.69–8.79)	<0.0001
Adenovírus (40/41)	9/1044 (1)	12/493 (2)	3.05 (1.25–7.75)	0.0144	4.43 (1.62–12.57)	0.0038
Nontyphoidal Salmonella	6/1047 (1)	6/495 (1)	2.53 (0.81–7.85)	0.108
Campylobacter	52/1047 (5)	30/495 (6)	1.45 (0.88–2.38)	0.1456
Sapovírus	29/1047 (3)	9/495 (2)	0.61 (0.26–1.28)	0.1967
Enteroaggregative E. coli (aaic only)	59/1047 (6)	40/495 (8)	1.38 (0.89–2.12)	0.1527
12–23 months
Shigella spp.	2/629 (0)	21/276 (8)	20.75 (6.61–103.57)	<0.0001	20.6 (6.30–105.59)	<0.0001
Cryptosporidium	60/628 (10)	51/275 (19)	2.63 (1.70–4.08)	<0.0001	2.61 (1.62–4.23)	<0.0001
Giardia	278/628 (44)	74/275 (27)	0.44 (0.32–0.61)	<0.0001	045 (0.32–0.63)	<0.0001
Rotavirus	102/628 (16)	62/275 (23)	1.87 (1.28–2.75)	0.0014	2.07 (1.36–3.16)	0.0007
EAEC (aatA only)	30/629 (5)	30/276 (11)	2.14 (1.22–3.80)	0.0079	2.12 (1.15–3.96)	0.0168
Aeromonas spp.	2/629 (0)	3/276 (1)	4.20 (0.82–25.21)	0.0837
Vibrio cholerae 01	1/629 (0)	4/276 (1)	3.97 (0.69–41.39)	0.1255
Nontyphoidal Salmonella	0/629 (0)	2/276 (1)	15.00 (1.22–2068.84)	0.0336
Adenovírus (40/41)	12/628 (2)	4/272 (1)	1.08 (0.33–2.97)	0.1416
24–59 months
Shigella spp.	1/303 (0)	29/145 (20)	126.56 (17.67–16063.77)	<0.0001	191 (23.80–24565.84)	<0.0001
Giardia	155/303 (51)	50/145 (34)	0.51 (0.32–0.78)	0.0022	0.43 (0.25–0.72)	0.001
EAEC (aatA/aaic)	10/303 (3)	1/145 (1)	0.25 (0.03–1.13)	0.0743	0.07 (0.00–0.81)	0.0284
V. cholerae	0/303 (0)	9/145 (6)	23.41 (2.87–3044.31)	0.0008	19.6 (2.48–2523.83)	0.0015
Cryptosporidium	22/303 (7)	11/145 (8)	1.35 (0.60–2.94)	0.4577
Norovírus	15/303 (5)	5/145 (3)	0.46 (0.16–1.18)	0.1091
Adenovírus (non-40/41)	8/303 (3)	0/145 (0)	0.11 (0.00–1.01)	0.0519

Clinical presentation

The clinical characteristics (signs and symptoms) of children infected with Shigella were compared with non-MSD Shigella using crude and multivariate analysis and presented in Table 3. The odds of having Shigella infections were associated with significant clinical features according to age, and the results from adjusted analysis are highlighted here. Dysentery, a classic symptom of shigellosis, was one of the most important features of diarrhea caused by Shigella compared to non-Shigella-MSD in both children aged 12–23 months (aOR =3.99; 95% CI: 153–10.39; P=0.0046) and 24–59 months (aOR =5.29; 95% CI: 2.05–13.66; P=0.0006). Moreover, the proportion of Shigella isolation was high among bloody diarrheal cases (20.1%; 32/158) compared to only 3.2% (24/758) for watery diarrhea, with the highest rate in children aged 24–59 months accounting with approximately one-third of all bloody diarrheal cases (Table 4). Fever was also an important symptom of Shigella-MSD in children from 12 to 23 months (aOR =3.02; 1.15–7.94; P=0.025) while rectal prolapse in 24–59 months (aOR =32.08; 1.18–869.71; P=0.0394). Irritability or restless attitude was an important sign for infants by crude analysis (OR =4.93; 1.10–22.9; P=0.0372).

Table 3

Clinical presentation of Shigella-associated MSD by crude and pathogen-adjusted analysis

Viables	Type of MSD crude analysis				Adjusted analysis
	Non-Shigella-MSD, n (%)	Shigella-MSD, n (%)	OR (95% CI)	P-value	Adjusted OR (95% CI)	P-value
0–11 months of age
Dysentery	35/489 (7)	1/6 (17)	3.49 (0.56–21.93)	0.1823
Sunken eyes	279/489 (57)	3/6 (50)	0.75 (0.17–3.35)	0.7096
Loss of skin turgor	172/489 (35)	2/6 (33)	1.02 (0.22–4.85)	0.9776
Intravenous rehydration	259/489 (53)	3/6 (50)	0.89 (0.20–3.95)	0.8763
Hospitalized	344/489 (70)	5/6 (83)	1.55 (0.25–9.52)	0.6369
Vomiting 3 or more times per daya	266/489 (54)	2/6 (33)	0.47 (0.10–2.21)	0.3361
Very thirstya	345/487 (71)	4/6 (67)	0.74 (0.16–3.53)	0.7079
Drank much less than usuala	70/489 (14)	1/6 (17)	1.62 (0.26–10.05)	0.6027
Unable to drinka	43/489 (9)	0/6 (0)	0.79 (0.04–14.25)	0.8728
Belly paina	129/489 (26)	2/6 (33)	1.55 (0.33–7.35)	0.5836
Fevera	170/489 (35)	2/6 (33)	1.04 (0.22–4.94)	0.9596
Irritability or restless attitudea	82/488 (17)	3/6 (50)	4.93 (1.10–22.09)	0.0372
Decreased activity or lethargya	238/488 (49)	1/6 (17)	0.29 (0.05–1.76)	0.1768
Loss of consciousnessa	8/489 (2)	0/6 (0)	4.36 (0.23–83.70)	0.3290
Rectal straininga	24/489 (5)	0/6 (0)	1.46 (0.08–26.70)	0.7979
Rectal prolapsea	2/489 (0)	0/5 (0)	17.73 (0.76–413.90)	0.0737
Cougha	331/489 (68)	3/6 (50)	0.48 (0.11–2.13)	0.3329
Difficulty breathinga	54/489 (11)	1/6 (17)	2.18 (0.35–13.55)	0.4034
Convulsiona	13/489 (3)	0/6 (0)	2.72 (0.15–50.69)	0.5036
Very thirstyb	349/487 (72)	4/6 (67)	0.71 (0.15–3.39)	0.6709
Drinks poorlyb	60/488 (12)	1/6 (17)	1.93 (0.31–11.99)	0.4796
Sunken eyesb	268/488 (55)	3/6 (50)	0.82 (0.18–3.65)	0.7959
Wrinkled skinb	165/488 (34)	1/6 (17)	0.53 (0.09–3.27)	0.4970
Irritable or restlessb	78/489 (16)	2/6 (33)	2.91 (0.61–13.93)	0.1806
Lethargy or loss of consciousnessb	238/489 (49)	1/6 (17)	0.29 (0.05–1.76)	0.1782
Dry mouthb	216/488 (44)	1/6 (17)	0.34 (0.06–2.11)	0.2480
Fast breathingb	148/489 (30)	1/6 (17)	0.63 (0.10–3.85)	0.6145
Undernutrition	48/489 (10)	0/6 (0)	0.70 (0.04–12.62)	0.8092
Low or very low skin pinch	144/450 (32)	1/5 (20)	0.71 (0.11–4.53)	0.7147
12–23 months of age
Dysentery	45/255 (18)	9/21 (43)	3.52 (1.43–8.67)	0.0063	3.99 (1.53–10.39)	0.0046
Sunken eyes	121/255 (47)	6/21 (29)	0.46 (0.18–1.20)	0.1127
Loss of skin turgor	66/255 (26)	4/21 (19)	0.73 (0.25–2.14)	0.5700
Intravenous rehydration	113/255 (44)	7/21 (33)	0.65 (0.26–1.62)	0.3553
Hospitalized	172/255 (67)	12/21 (57)	0.64 (0.26–1.54)	0.3168
Vomiting 3 or more times per daya	124/255 (49)	7/21 (33)	0.55 (0.22–1.36)	0.1953
Very thirstya	208/255 (82)	15/20 (75)	0.64 (0.23–1.79)	0.3957
Drank much less than usuala	14/255 (5)	1/21 (5)	1.22 (0.21–6.95)	0.8238
Unable to drinka	16/255 (6)	1/21 (5)	1.06 (0.19–6.00)	0.9456
Belly paina	68/255 (27)	6/21 (29)	1.15 (0.44–2.99)	0.7776
Fevera	84/255 (33)	10/21 (48)	1.85 (0.77–4.45)	0.1676	3.02 (1.15–7.94)	0.025
Irritability or restless attitudea	40/255 (16)	1/21 (5)	0.39 (0.07–2.11)	0.2744
Decreased activity or lethargya	135/255 (53)	9/21 (43)	0.68 (0.28–1.63)	0.3821
Loss of consciousnessa	5/255 (2)	0/21 (0)	1.06 (0.06–19.80)	0.9693
Rectal straininga	12/254 (5)	1/21 (5)	1.42 (0.25–8.19)	0.6952
Rectal prolapsea	0/255 (0)	0/21 (0)	–	–
Cougha	159/255 (62)	7/21 (33)	0.31 (0.13–0.78)	0.0130	0.27 (0.10–0.72)	0.0085
Difficulty breathinga	21/255 (8)	0/21 (0)	0.25 (0.01–4.33)	0.3435
Convulsiona	12/255 (5)	1/21 (5)	1.43 (0.25–8.22)	0.6918
Very thirstyb	209/255 (82)	15/21 (71)	0.53 (0.20–1.40)	0.1983
Drinks poorlyb	15/254 (6)	1/21 (5)	1.13 (0.20–6.41)	0.8897
Sunken eyesb	124/255 (49)	6/21 (29)	0.44 (0.17–1.14)	0.0923
Wrinkled skinb	62/255 (24)	2/21 (10)	0.40 (0.10–1.53)	0.1788
Irritable or restlessb	41/255 (16)	2/21 (10)	0.66 (0.17–2.58)	0.5526
Lethargy or loss of consciousnessb	125/253 (49)	9/21 (43)	0.78 (0.32–1.87)	0.5759
Dry mouthb	112/255 (44)	8/21 (38)	0.80 (0.33–1.96)	0.6304
Fast breathingb	53/253 (21)	2/21 (10)	0.48 (0.12–1.85)	0.2875
Undernutrition	69/255 (27)	4/21 (19)	0.69 (0.24–2.02)	0.4975
Low or very low skin pinch	50/237 (21)	4/19 (21)	1.08 (0.36–3.22)	0.8931
24–59 months of age
Dysentery	46/116 (40)	22/29 (76)	4.55 (1.84–11.25)	0.0010	5.29 (2.05–13.66)	0.0006
Sunken eyes	43/116 (37)	8/29 (28)	0.67 (0.28–1.61)	0.3679
Loss of skin turgor	20/116 (17)	2/29 (7)	0.43 (0.11–1.70)	0.2279
Intravenous rehydration	34/116 (29)	5/29 (17)	0.54 (0.20–1.47)	0.2258
Hospitalized	59/116 (51)	5/29 (17)	0.22 (0.08–0.59)	0.0026
Vomiting 3 or more times per daya	38/116 (33)	3/29 (10)	0.27 (0.08–0.88)	0.0293
Very thirstya	93/116 (80)	22/29 (76)	0.75 (0.29–1.93)	0.5569
Drank much less than usuala	11/116 (9)	1/29 (3)	0.48 (0.08–2.78)	0.4151
Unable to drinka	9/116 (8)	3/29 (10)	1.49 (0.41–5.47)	0.5438
Belly paina	23/116 (20)	10/29 (34)	2.14 (0.89–5.14)	0.0883
Fevera	40/115 (35)	11/29 (38)	1.16 (0.51–2.66)	0.7275
Irritable or restlessa	9/115 (8)	2/29 (7)	1.02 (0.24–4.37)	0.9796
Decreased activity or lethargya	58/115 (50)	10/29 (34)	0.53 (0.23–1.22)	0.1348
Loss of consciousnessa	2/116 (2)	1/29 (3)	2.41 (0.31–19.02)	0.4038
Rectal straininga	9/115 (8)	5/29 (17)	2.52 (0.81–7.86)	0.1120
Rectal prolapsea	0/116 (0)	1/29 (3)	12.26 (0.49–308.99)	0.1279	32.08 (1.18–869.71)	0.0394
Cougha	52/116 (45)	13/29 (45)	1.01 (0.45–2.25)	0.9900
Difficulty breathinga	2/115 (2)	0/29 (0)	0.77 (0.04–16.46)	0.8669
Convulsiona	8/116 (7)	1/29 (3)	0.67 (0.11–4.00)	0.6621
Very thirstyb	94/116 (81)	22/29 (76)	0.71 (0.28–1.84)	0.4856
Drinks poorlyb	15/116 (13)	1/29 (3)	0.34 (0.06–1.94)	0.2266
Sunken eyesb	45/116 (39)	8/29 (28)	0.62 (0.26–1.49)	0.2873
Wrinkled skinb	19/116 (16)	2/29 (7)	0.45 (0.11–1.81)	0.2638
Irritability or restless attitudeb	3/116 (3)	2/29 (7)	2.95 (0.55–15.75)	0.2060
Lethargy or loss of consciousnessb	52/116 (45)	9/29 (31)	0.57 (0.24–1.33)	0.1946
Dry mouthb	35/116 (30)	5/29 (17)	0.52 (0.19–1.41)	0.1962
Fast breathingb	8/116 (7)	0/29 (0)	0.22 (0.01–3.86)	0.2977
Undernutrition	8/116 (7)	0/29 (0)	0.22 (0.01–3.86)	0.2977
Low or very low skin pinch	16/108 (15)	1/28 (4)	0.31 (0.05–1.72)	0.1782

Notes:

Since began;

current.

Abbreviation: MSD, moderate-to-severe diarrhea.

Table 4

Crude analysis of Shigella-associated diarrhea by severity (bloody diarrhea and watery diarrhea) in the different age groups (December 2007–November 2012)

Age groups	Watery diarrhea, n (%)	Bloody diarrhea, n (%)	OR (95% CI)	P-value
0–11 months	5/459 (1)	1/36 (3)	3.49 (0.56–21.93)	0.1823
12–23 months	12/222 (5)	9/54 (17)	3.52 (1.43–8.67)	0.0063
24–59 months	7/77 (9)	22/68 (32)	4.55 (1.84–11.25)	0.001

Risk factors

The role of different risk factors for MSD was age-dependent, although most of the studied socioeconomic or hygienic explanatory variables were not statistically significant (Table 5). Partial or exclusive breastfeeding was a remarkable protective factor against Shigella in younger children aged 0–11 months with MSD by crude analysis (OR =0.10; 0.02–0.68; P=0.0184). Protective factors in children aged 12–23 months included partial or exclusive breastfeeding (OR =0.12; 0.04–0.40; P=0.0006), mother as primary child caretaker (aOR =0.28; 0.09–0.87; P=0.0281), and giving stored water to child (aOR =0.17; 0.04–0.82; P=0.0267). In 24–59 months, water availability (not always per day) (aOR =0.32; 0.12–0.81; P=0.0167) and washing hands before preparing baby’s food (aOR =0.28; 0.08–0.94; P=0.0394) were protective against shigellosis. Among controls (Table 6), relevant protective factors for acquiring Shigella included child primary caretaker for children aged 0–11 months (aOR =0.03; 0.00–0.87; P=0.0408), and washing hands before eating in children aged 12–23 months (aOR =0.05; 0.01, 0.52; P=0.0119).

Table 5

Risk factors of Shigella-associated moderate-to-severe diarrhea by crude and adjusted analysis

Variables	Shigella spp.		Crude analysis		Adjusted analysis
	Negative, n (%)	Positive, n (%)	OR (95% CI)	P-value	Adjusted OR (95% CI)	P-value
0–11 months
>6 months of age	259/489 (53)	5/6 (83)	3.26 (0.53–19.98)	0.2021
Child sex (male)	294/489 (60)	4/6 (67)	1.19 (0.25–5.67)	0.8226
Child primary caretaker (mother)	473/489 (97)	6/6 (100)	0.45 (0.02–8.38)	0.5948
Caretaker formal education	135/487 (28)	3/6 (50)	2.60 (0.58–11.60)	0.2100
Animals in compound	411/489 (84)	4/6 (67)	0.34 (0.07–1.64)	0.1806
Water availability (not always per day)	256/488 (52)	4/6 (67)	1.63 (0.34–7.73)	0.5375
Access to improved water	411/489 (84)	4/6 (67)	0.34 (0.07–1.64)	0.1806
Give stored water to child	450/489 (92)	6/6 (100)	1.14 (0.06–20.61)	0.9294
Treating water habit	54/489 (11)	1/6 (17)	2.18 (0.35–13.55)	0.4034
Facility to dispose child’s stool	248/486 (51)	2/6 (33)	0.53 (0.11–2.53)	0.4284
Improved facility for household stool	43/488 (9)	0/6 (0)	0.79 (0.04–14.22)	0.8716
Wash hands before eating	463/489 (95)	5/6 (83)	0.21 (0.03–1.33)	0.0974
Wash hands before cooking	304/489 (62)	3/6 (50)	0.61 (0.14–2.71)	0.5152
Wash hands before preparing baby’s food	129/489 (26)	3/6 (50)	2.78 (0.62–12.42)	0.1796
Wash hands after defecating	413/489 (84)	6/6 (100)	2.41 (0.13–43.14)	0.5513
Wash hands after handling animals	12/489 (2)	0/6 (0)	2.94 (0.16–55.07)	0.4710
Wash hands after cleaning child feces	127/489 (26)	2/6 (33)	1.58 (0.33–7.51)	0.5655
Partial or exclusive breastfeeding	413/424 (97)	5/6 (83)	0.10 (0.02–0.68)	0.0184
Lowest quintile of wealth index	110/484 (23)	2/6 (33)	1.88 (0.40–8.96)	0.4268
12–23 months
>18 months of age	75/255 (29)	10/21 (48)	2.18 (0.91–5.26)	0.0816
Child sex (male)	147/255 (58)	10/21 (48)	0.67 (0.28–1.61)	0.3713
Child primary caretaker (mother)	237/255 (93)	17/21 (81)	0.30 (0.10–0.95)	0.0398	0.28 (0.09–0.87)	0.0281
Caretaker formal education	58/253 (23)	4/21 (19)	0.86 (0.29–2.52)	0.7825
Animals in compound	213/255 (84)	16/21 (76)	0.60 (0.22–1.66)	0.3217
Water availability (not always per day)	140/255 (55)	11/21 (52)	0.90 (0.38–2.15)	0.8135
Access to improved water	214/255 (84)	16/21 (76)	0.58 (0.21–1.61)	0.2962
Give stored water to child	249/255 (98)	19/21 (90)	0.20 (0.04–0.94)	0.0412	0.17 (0.04–0.82)	0.0267
Treating water habit	14/255 (5)	2/21 (10)	2.14 (0.52–8.83)	0.2948
Facility to dispose child’s stool	207/253 (82)	18/21 (86)	1.18 (0.36–3.88)	0.7796
Improved facility for household stool	15/255 (6)	0/21 (0)	0.36 (0.02–6.24)	0.4834
Wash hands before eating	225/255 (88)	19/21 (90)	1.05 (0.27–4.15)	0.9390
Wash hands before cooking	152/255 (60)	11/21 (52)	0.74 (0.31–1.78)	0.5055
Wash hands before preparing baby’s food	78/255 (31)	4/21 (19)	0.58 (0.20–1.69)	0.3203
Wash hands after defecating	219/255 (86)	19/21 (90)	1.30 (0.33–5.07)	0.7083
Wash hands after handling animals	4/255 (2)	0/21 (0)	1.30 (0.07–24.95)	0.8619
Wash hands after cleaning child feces	59/255 (23)	7/21 (33)	1.71 (0.68–4.32)	0.2581
Partial or exclusive breastfeeding	139/214 (65)	3/18 (17)	0.12 (0.04–0.40)	0.0006
Lowest quintile of wealth index	55/255 (22)	2/21 (10)	0.46 (0.12–1.79)	0.2637
24–59 months
>42 months of age	25/116 (22)	3/29 (10)	0.47 (0.14–1.57)	0.2218
Child sex (male)	68/116 (59)	19/29 (66)	1.31 (0.57–3.03)	0.5208
Child primary caretaker (mother)	100/116 (86)	28/29 (97)	3.12 (0.56–17.46)	0.1955
Caretaker formal education	23/115 (20)	11/29 (38)	2.45 (1.03–5.81)	0.0424
Animals in compound	95/116 (82)	24/29 (83)	1.00 (0.36–2.83)	0.9957
Water availability (not always per day)	60/116 (52)	7/29 (24)	0.31 (0.13–0.77)	0.0113	0.32 (0.12–0.81)	0.0167
Access to improved water	95/116 (82)	25/29 (86)	1.28 (0.42–3.86)	0.6661
Give stored water to child	109/115 (95)	27/29 (93)	0.65 (0.14–2.98)	0.5819
Treating water habit	5/116 (4)	3/29 (10)	2.68 (0.66–10.92)	0.1696
Facility to dispose child’s stool	110/116 (95)	28/29 (97)	1.12 (0.18–6.92)	0.9048
Improved facility for household stool	13/116 (11)	4/29 (14)	1.35 (0.43–4.28)	0.6069
Wash hands before eating	106/116 (91)	27/29 (93)	1.08 (0.26–4.58)	0.9122
Wash hands before cooking	70/116 (60)	19/29 (66)	1.22 (0.53–2.83)	0.6346
Wash hands before preparing baby’s food	34/116 (29)	3/29 (10)	0.32 (0.10–1.03)	0.0563	0.28 (0.08–0.94)	0.0394
Wash hands after defecating	101/116 (87)	24/29 (83)	0.68 (0.23–1.98)	0.4796
Wash hands after handling animals	4/116 (3)	1/29 (3)	1.32 (0.20–8.74)	0.7763
Wash hands after cleaning child feces	23/116 (20)	6/29 (21)	1.10 (0.41–2.93)	0.8480
Partial or exclusive breastfeeding	18/100 (18)	4/20 (20)	1.22 (0.38–3.87)	0.7404
Lowest quintile of wealth index	31/115 (27)	7/29 (24)	0.89 (0.36–2.25)	0.8121

Table 6

Risk factors for Shigella infection in controls by crude and adjusted analysis

Variables	Shigella spp.		Crude analysis		Adjusted analysis
	Negative, n (%)	Positive, n (%)	OR (95% CI)	P-value	Adjusted OR (95% CI)	P-value
0–11 months
>6 months of age	595/1046 (57)	1/1 (100)	2.27 (0.09–55.97)	0.6151
Child sex (male)	629/1046 (60)	1/1 (100)	1.99 (0.08–48.96)	0.6738
Child primary caretaker (mother)	1035/1046 (99)	1/1 (100)	0.03 (0.00–0.86)	0.0404	0.03 (0.00–0.87)	0.0408
Caretaker formal education	313/1044 (30)	1/1 (100)	7.00 (0.28–172.30)	0.2338
Animals in compound	897/1045 (86)	1/1 (100)	0.50 (0.02–12.24)	0.6684
Water availability (not always per day)	333/1046 (32)	0/1 (0)	0.71 (0.03–17.55)	0.8361
Access to improved water	888/1046 (85)	0/1 (0)	0.06 (0.00–1.47)	0.0844
Give stored water to child	829/1046 (79)	0/1 (0)	0.09 (0.00–2.15)	0.1360
Treating water habit	66/1046 (6)	0/1 (0)	4.91 (0.20–121.81)	0.3310
Facility to dispose child’s stool	656/1046 (63)	1/1 (100)	1.78 (0.07–43.91)	0.7231
Improved facility for household stool	79/1046 (8)	0/1 (0)	4.06 (0.16–100.39)	0.3924
Wash hands before eating	980/1046 (94)	1/1 (100)	0.20 (0.01–5.04)	0.3310
Wash hands before cooking	855/1046 (82)	0/1 (0)	0.07 (0.00–1.84)	0.1124
Wash hands before preparing baby’s food	627/1046 (60)	1/1 (100)	2.01 (0.08–49.35)	0.6702
Wash hands after defecating	765/1046 (73)	1/1 (100)	1.10 (0.04–27.16)	0.9521
Wash hands after handling animals	421/1046 (40)	0/1 (0)	0.49 (0.02–12.17)	0.6667
Wash hands after cleaning child feces	642/1046 (61)	1/1 (100)	1.89 (0.08–46.48)	0.6972
Partial or exclusive breastfeeding	1018/1039 (98)	1/1 (100)	0.06 (0.00–1.60)	0.0939
Lowest quintile of wealth index	194/1045 (19)	0/1 (0)	1.46 (0.06–35.96)	0.8172
12–23 months
>18 months of age	200/627 (32)	0/2 (0)	0.43 (0.02–8.92)	0.5828
Child sex (male)	373/627 (59)	2/2 (100)	3.41 (0.16–71.26)	0.4294
Child primary caretaker (mother)	598/627 (95)	2/2 (100)	0.25 (0.01–5.25)	0.3695
Caretaker formal education	172/625 (28)	1/2 (50)	2.63 (0.27–25.45)	0.4039
Animals in compound	555/627 (89)	2/2 (100)	0.65 (0.03–13.73)	0.7836
Water availability (not always per day)	216/627 (34)	2/2 (100)	9.50 (0.45–198.84)	0.1467
Access to improved water	521/627 (83)	1/2 (50)	0.20 (0.02–1.98)	0.1707
Give stored water to child	583/625 (93)	2/2 (100)	0.36 (0.02–7.71)	0.5166
Treating water habit	26/627 (4)	0/2 (0)	4.54 (0.21–96.93)	0.3327
Facility to dispose child’s stool	600/626 (96)	2/2 (100)	0.22 (0.01–4.71)	0.3333
Improved facility for household stool	27/627 (4)	0/2 (0)	4.37 (0.20–93.17)	0.3451
Wash hands before eating	596/627 (95)	1/2 (50)	0.05 (0.01–0.52)	0.0119	0.05 (0.01–0.52)	0.0119
Wash hands before cooking	522/627 (83)	2/2 (100)	1.01 (0.05–21.18)	0.9951
Wash hands before preparing baby’s food	386/627 (62)	0/2 (0)	0.12 (0.01–2.61)	0.1801
Wash hands after defecating	454/627 (72)	2/2 (100)	1.91 (0.09–39.96)	0.6770
Wash hands after handling animals	254/627 (41)	0/2 (0)	0.29 (0.01–6.14)	0.4294
Wash hands after cleaning child feces	367/627 (59)	0/2 (0)	0.14 (0.01–2.97)	0.2079
Partial or exclusive breastfeeding	443/621 (71)	2/2 (100)	2.01 (0.10–42.13)	0.6522
Lowest quintile of wealth index	135/627 (22)	1/2 (50)	3.63 (0.38–35.22)	0.2654
24–59 months
>42 months of age	51/302 (17)	0/1 (0)	1.63 (0.07–40.52)	0.7664
Child sex (male)	201/302 (67)	0/1 (0)	0.17 (0.01–4.16)	0.2759
Child primary caretaker (mother)	268/302 (89)	1/1 (100)	0.39 (0.02–9.65)	0.5618
Caretaker formal education	74/302 (25)	0/1 (0)	1.02 (0.04–25.37)	0.9892
Animals in compound	252/302 (83)	1/1 (100)	0.60 (0.02–14.94)	0.7555
Water availability (not always per day)	103/302 (34)	0/1 (0)	0.64 (0.03–15.91)	0.7870
Access to improved water	254/302 (84)	1/1 (100)	0.57 (0.02–14.24)	0.7332
Give stored water to child	277/302 (92)	1/1 (100)	0.28 (0.01–6.94)	0.4337
Treating water habit	9/302 (3)	0/1 (0)	10.30 (0.39–269.66)	0.1616
Facility to dispose child’s stool	298/301 (99)	1/1 (100)	0.04 (0.00–1.02)	0.0515
Improved facility for household stool	24/302 (8)	0/1 (0)	3.79 (0.15–95.51)	0.4185
Wash hands before eating	286/302 (95)	1/1 (100)	0.17 (0.01–4.41)	0.2880
Wash hands before cooking	232/302 (77)	1/1 (100)	0.91 (0.04–22.58)	0.9539
Wash hands before preparing baby’s food	156/302 (52)	0/1 (0)	0.31 (0.01–7.72)	0.4768
Wash hands after defecating	210/302 (70)	0/1 (0)	0.15 (0.01–3.63)	0.2408
Wash hands after handling animals	122/302 (40)	1/1 (100)	4.42 (0.18–109.40)	0.3640
Wash hands after cleaning child feces	161/302 (53)	1/1 (100)	2.63 (0.11–65.04)	0.5550
Partial or exclusive breastfeeding	8/279 (3)	0/1 (0)	10.65 (0.40–280.87)	0.1566
Lowest quintile of wealth index	53/302 (18)	0/1 (0)	1.55 (0.06–38.68)	0.7879

Impact of antibiotic treatment on patient outcome

The impact of antibiotic treatment on patient outcome was assessed among children with known outcome (Table 7). Patient outcome was available for all of Shigella-diarrheal children while antibiotic treatment was administered in only 30.1% (22/73). The most administered antibiotics were ampicillin and gentamicin (12.3%; 9/73), followed by trimethoprim-sulfametoxazole (9.6%; 7/73), chloramphenicol (8.2%; 6/73), and nalidixic acid (6.9%; 5/73). Although high rates of resistance were found, patient improvement at discharge was much higher in children that received antibiotic treatment (63.0%; 17/27) compared to 37.0% (10/27), for those without any treatment (P<0.0001).

Table 7

Impact of antibiotic treatment for Shigella-diarrhea in children outcome when leaving the hospital from December 2007 to November 2012

Outcomes	Antibiotic treatment, n (%)
	No	Yes	Total	P-value
Improved	10/27 (37)	17/27 (63)	27	<0.0001
Not improved	41/46 (89.1)	5/46 (10.8)	46
Total	51/73 (69.8)	22/73 (30.1)	73
Antibiotics administered	Proportion, n (%)		Resistance, n (%)
Trimethoprim-sulfametoxazole	7/73 (9.6)		62/67 (92.5)
Gentamicin	9/73 (12.3)		0
Chloramphenicol	6/73 (8.2)		36/67 (53.7)
Amoxicillin clavulanic acid	4/73 (5.5)		3/67 (4.5)
Ampicillin	9/73 (12.3)		34/67 (50.7)
Nalidixic acid	5/73 (6.9)		0
Ciprofloxacin	1/73 (1.4)		0

Discussion

In this prospective, case–control study, we demonstrated the association of Shigella spp. with MSD and LSD among children under 5 years in Manhiça District, a rural area of southern Mozambique. Its low detection rate in children without diarrhea and in cases of LSD confirms the high probability that whenever present, this pathogen causes a virulent disease, which could be explained in part by the low infectious dose of the bacterium (10–100 cells).21 While an association between Enterobacteriaceae and Giardia has been reported,22 in this study we showed that the prevalence of Giardia was independent of Shigella by adjusted analysis. In addition, Giardia was more common in controls while Shigella was mostly isolated in cases. The proportion of cases presenting with Shigella was similar with those reported in our previous study12 and in other African countries.3,23,24 Overall, the detection rate of Shigella was high during rainy season, which is similar to other studies.25 As expected, the classical signs/symptoms of Shigella-MSD were mostly of dysentery accompanied with fever or rectal prolapse.8 Irritability or restless attitude was also an importance feature in infants. The observation that Shigella may also be associated with less severe cases is a matter of concern, because the disease is mostly considered severe and frequently associated with dysentery. These findings expand the spectrum of clinical features associated with Shigella infection and demonstrate the importance of active surveillance for disease prevention. Another explanation could be related to health-seeking behavior, meaning that mothers or child caretakers are looking for assistance earlier before the disease becomes severe, as demonstrated in our community study about health care utilization and attitudes in cases of MSD.26

The finding that children who received antibiotic treatment was associated with a good outcome at the discharge, reinforcing the importance of antibiotic therapy for the management of shigellosis. Similar reports have shown that, with an effective antibiotic therapy, clinical improvement occurs within 48 hours,27 resulting in a decreased risk of serious complications and death, shorter duration of symptoms, the elimination of Shigella from the stool, and subsequently decreased infection transmission.

MSD caused by Shigella was associated with a high OR compared to other significant pathogens in adjusted analysis, which may suggest its high pathogenicity as observed by the low detection rate in healthy children compared to other significant pathogens. In accordance with previous reports,28 we found a low risk of Shigella-associated diarrhea in infancy and a steady increase through the second year of life. One likely explanation is that the partial or exclusive breastfeeding, which was present in 97% of infants (<12 months) of age and in 61% of children under 2 years in our study, neutralized the infectious inoculum and prevented disease. The strong association between MSD caused by Shigella and children over 2 years of age has been attributed to a decreased exposure to breast milk and an increased exposure to sources of Shigella infection such as hands contact in the environment as children become more active.29,30 In addition, the benefits of human milk for infant health have been recognized as an important source of bacteria that may contribute to neonatal gastrointestinal colonization leading to immune response development and possible infection prevention. Thus, the microbiota of breast-fed infants is considered the gold standard in terms of a healthy infant gastrointestinal microbiota.31

In addition, as children wean, they are exposed to an increased array of food and water that could serve as sources of Shigella, thus increasing the probability of getting infected.32 Therefore, this study also sought to quantify the effects of several socioeconomic indicators on the relative increased risk for acquiring Shigella in children presenting MSD. Overall, the role of the assessed risk factors for Shigella-MSD was age-related, possibly due to differences in the degree of exposure of the studied population. The presence of broader protective factors for diarrhea caused by Shigella in the present study could be attributed to an improved health-seeking behavior among the cases and improvement in accessibility of health services over the years. The population leaving in Manhiça district is under monitoring through HDSS since 1996, and is thus exposed to several interventions that contribute to health education that may influence the health-seeking behavior. This may explain why socioeconomic characteristics such as formal education were less relevant in the present study. Considering these variations, the importance of children primary caretaker (mother), giving stored water to child, water availability (not always per day), and washing hands before preparing baby’s food in preventing diarrhea caused by Shigella is highlighted.

The importance of hand-washing practices in preventing diarrheal illness is supported by a number of studies.33,34 A systematic review of several studies estimated that appropriate hand washing with soap could reduce the risks of severe intestinal infections and shigellosis up to 48% and 59%, respectively.34 The fact that stored water consumption was protective for Shigella infection leads us to believe that the hygienic conditions for water transportation and the containers where the water is stored are adequate or properly cleaned, because water contamination can occur at any stage from the source to the point of use. Conversely, unavailability of water throughout the day was protective for Shigella infection and may reflect the rational use of scarce water at the household level, or be linked to the hypothesis that the lack of water can interrupt the chain of transmission of some waterborne transmitted enteropathogens like Shigella as families are less exposed due to the limited amount of water that they have. The prevalence of malnutrition in both Shigella-associated MSD and in non-Shigella MSD highlights the vicious cycle of malnutrition and diarrhea. While malnutrition makes children more vulnerable to diarrheal infections due to its negative effect on the barrier protection leading to deterioration of the immune system, chronic infections can contribute to long-term gut damage and preventing nutrient absorption.35

Conclusion

Shigella is a significant pathogen associated with MSD in Mozambican children from 1 to 5 years. The presence of cases of LSD caused by Shigella is a matter of concern and may suggest the need to expand the clinical feature of the disease. The presence of dysentery remains an important clinical feature of Shigella-associated diarrhea. The study reinforces the recommendations from the WHO on the use of antibiotic treatment for the management of shigellosis while vaccines to prevent disease are not available. As demonstrated in this study, promoting awareness of good personal hygiene and a safe water storage is of great importance in prevention and control of the disease. Moreover, continuous surveillance studies are needed to track changes over time.