Abstract.
Shigella is a major cause of severe diarrhea in children less than the age of 5 years in sub-Saharan Africa. The aim of this study was to describe the (sub-)serotype distribution and antimicrobial susceptibility of Shigella serogroups from Centrafrican patients with diarrhea between 2002 and 2013. We collected 443 Shigella isolates in total. The most common serogroups were Shigella flexneri (N = 243, 54.9%), followed by Shigella sonnei (N = 90, 20.3%) and Shigella dysenteriae (N = 72, 16.3%). The high diversity of (sub-)serotypes of S. flexneri and S. dysenteriae may impede the development of an efficient vaccine. Rates of resistance were high for ampicillin, chloramphenicol, tetracycline, and cotrimoxazole but low for many other antimicrobials, confirming recommendations for the use of third-generation cephalosporins (only one organism resistant) and fluoroquinolones (no resistance). However, the detection of one extended-spectrum beta-lactamase–producing Shigella organism highlights the need for continued monitoring of antimicrobial drug susceptibility.
INTRODUCTION
In 2013, 6.3 million deaths were recorded in children less than the age of 5 years, 578,000 of which were because of diarrheal disease, the second leading cause of death in this age group. Almost half of these diarrhea-related deaths occurred in sub-Saharan Africa.1 Recent studies (Torcadia for Central African Republic [CAR] and the Global Enteric Multicenter Study for other sites) in The Gambia and Mali (West Africa), Mozambique and Kenya (East Africa), and CAR (Central Africa) have confirmed the continuing importance of Shigella as a major cause of severe diarrhea in children less than the age of 5 years. Shigella spp. are the third most important pathogen in these regions.2,3
High rates of resistance to conventional antimicrobials, such as ampicillin, chloramphenicol, tetracycline, and cotrimoxazole, have been reported for Shigella spp. in many studies.4 This resistance has led to third-generation cephalosporins (C3G), fluoroquinolones, and azithromycin becoming the first-line antimicrobials for treating these infections. However, the clinical severity of shigellosis and the emergence of resistance to first-line treatments highlight the growing need to develop alternative prophylactic and therapeutic strategies. The development of a safe and effective anti-Shigella vaccine for controlling shigellosis is enshrined in WHO public health policy. However, the presence of four different serogroups (formerly known as species), Shigella flexneri, Shigella dysenteriae, Shigella boydii, and Shigella sonnei, made up of at least 50 antigenically different (sub-)serotypes may impede vaccine development.5 Indeed, the larger the number of serotypes to be included, the more complex and expensive the vaccine becomes. The lack of data from Central Africa and from very low-income countries, such as CAR,6 which suffers from long-term instability, highlights the need to improve our understanding of the spatial and temporal distribution of (sub-)serotypes in sub-Saharan Africa.7,8 Central African Republic is a resource-limited country in equatorial Africa (ranked 180/187 according to the Human Development Index in 2013). Here, we describe the (sub-)serotype distribution and antimicrobial susceptibility of Shigella species isolated from patients in CAR during the 2002–2013 period.
Clinical isolates of Shigella were obtained between January 2002 and December 2013, from Centrafrican outpatients with diarrhea attending the Institut Pasteur in Bangui. If more than one isolate of the same (sub-)serotype and serogroup and with the same antimicrobial drug resistance phenotype was recovered from a given patient, only the first isolate was included. Shigella was identified by conventional methods and (sub-)serotyping was performed by slide agglutination assays with a complete set of antisera recognizing all the described Shigella serotypes.9 Antimicrobial drug susceptibility was assessed by the disk diffusion method, and extended-spectrum beta-lactamase (ESBL) production was evaluated in the double-disc synergy test, as previously described.10
Date, site of isolation, patient age, and gender were recorded for each isolate. We used χ2 test, Student’s t-test, and ANOVA (analysis of variance—with Lilliefors’ test for normality and Levene’s test for homoscedasticity) to compare categorical and continuous variables in univariate analysis. Multivariate logistic regression was performed to explore high rates of multidrug resistance (MDR, defined by resistance to more than five of the 13 antimicrobials tested). Factors with P values < 0.2 in univariate analysis were retained for the multivariate analysis. We considered P values < 0.05 to be statistically significant.
In total, 443 clinical isolates of Shigella were collected between January 2002 and December 2013, from 443 Centrafrican outpatients with diarrhea (238 male and 205 female patients, mean age: 27.2 years, median age: 29.5 years, 25th percentile: 8 years, 75th percentile: 40 years). The small number of organisms isolated during the study period reflects the poor access to health-care services in CAR, particularly for the laboratory diagnosis of diarrhea. Significant differences in the number of isolates obtained were also observed between years because of the economic and political crisis that occurred during the study period, further restricting patient access to health-care facilities. However, the distribution of serogroups recovered in our study for 2004–2005 was consistent with that reported for the same period in a study conducted at four health-care centers in Bangui.6 The data reported here may, therefore, be considered representative of the global situation in Bangui.
The incidence of S. dysenteriae infection has been reported to be higher in men than in women in China.11 By contrast, we found a significant association between S. dysenteriae infection and being female (odds ratio = 1.86 95% confidence interval [1.11–3.12]; P = 0.018). No significant association was found between gender and the other serogroups or between Shigella serogroup and age. Contrary to several other reports,11,12 we observed no significant seasonality in the distribution of Shigella isolates or in serogroup distribution (i.e., erratic variation between months, but no difference between the wet and dry seasons).
The most common serogroup was S. flexneri (N = 243, 54.9%), consistent with several reports from developing countries in Africa and Asia,5,13 followed by S. sonnei (N = 90, 20.3%) and S. dysenteriae (N = 72, 16.3%). Shigella boydii (N = 34, 7.7%), which is generally restricted to North and East Africa (Ethiopia and Egypt) and South Asia (Bangladesh and Nepal),13 was rarely encountered in our study (Table 1). Unsurprisingly, no significant difference in the prevalence of Shigella serogroups was observed during the study period, except for S. sonnei (P < 0.001). However, the prevalence of the S. sonnei serogroup fluctuated over time, with no significant trend (Table 1). This finding is consistent with the known distribution of Shigella serogroups in countries with a low socioeconomic level,4 CAR being one of the poorest countries of sub-Saharan Africa.
Table 1.
Distribution of the most frequent Shigella serogroups and (sub-)serotypes in Bangui, Central African Republic, 2002–2013
| Shigella | 2002 | 2003 | 2004 | 2005 | 2006 | 2007 | 2008 | 2009 | 2010 | 2011 | 2012 | 2013 | Total | P |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Serogroups/serotypes | N = 14 | N = 22 | N = 59 | N = 60 | N = 82 | N = 51 | N = 26 | N = 11 | N = 8 | N = 39 | N = 44 | N = 27 | N = 443 | |
| n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | ||
| flexneri* | 8 (57.1) | 14 (63.6) | 34 (57.6) | 34 (56.7) | 49 (59.8) | 32 (62.7) | 9 (34.6) | 5 (45.4) | 5 (62.5) | 18 (46.2) | 22 (50.0) | 13 (48.1) | 243 (54,9) | NS |
| 1 | 0 | 0 | 0 | 0 | 1 (1.2) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (0.2) | NS |
| 1a | 0 | 0 | 0 | 0 | 1 (1.2) | 0 | 0 | 0 | 1 (12.5) | 0 | 1 (2.3) | 0 | 3 (0.7) | 0.03 |
| 1b | 0 | 5 (22.7) | 11 (18.7) | 13 (21.7) | 15 (18.3) | 5 (9.8) | 3 (11.5) | 0 | 2 (25.0) | 2 (5.1) | 2 (4.5) | 1 (3.7) | 59 (13.3) | 0.03 |
| 1c | 1 (7.1) | 0 | 6 (10.2) | 0 | 0 | 2 (3.9) | 0 | 0 | 0 | 0 | 0 | 0 | 9 (2.0) | 0.002 |
| 2a | 7 (50.0) | 5 (22.7) | 3 (5.1) | 8 (13.3) | 3 (3.7) | 6 (11.8) | 0 | 1 (9.1) | 0 | 1 (2.6) | 6 (13.6) | 0 | 40 (9.0) | < 0.001 |
| 3a | 0 | 0 | 7 (11.9) | 2 (3.3) | 7 (8.5) | 8 (15.7) | 3 (11.5) | 1 (9.1) | 1 (12.5) | 2 (5.1) | 3 (6.8) | 4 (14.8) | 38 (8.6) | NS |
| 3b | 0 | 0 | 0 | 0 | 1 (1.2) | 0 | 0 | 0 | 0 | 0 | 1 (2.3) | 0 | 2 (0.5) | NS |
| 4a | 0 | 1 (4.5) | 0 | 0 | 3 (3.7) | 7 (13.7) | 2 (7.7) | 1 (9.1) | 0 | 2 (5.1) | 5 (11.4) | 4 (14.8) | 25 (5.6) | 0.02 |
| 4c | 0 | 0 | 2 (3.4) | 4 (6.7) | 2 (2.4) | 1 (2.0) | 0 | 0 | 0 | 1 (2.6) | 0 | 0 | 10 (2.3) | NS |
| 6a | 0 | 3 (13.6) | 5 (8.5) | 7 (11.7) | 15 (18.3) | 3 (5.9) | 1 (3.8) | 2 (18.2) | 1 (12.5) | 10 (25.6) | 4 (9.1) | 3 (11.1) | 64 (14.4) | NS |
| 7 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (3.7) | 1 (0.2) | NS |
| 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (12.5) | 0 | 0 | 0 | 1 (0.2) | NS |
| 9 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (3.8) | 0 | 0 | 0 | 0 | 0 | 1 (0.2) | NS |
| y | 0 | 0 | 0 | 0 | 1 (1.2) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (0.2) | NS |
| Prov93-119 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (3.7) | 1 (0.2) | NS |
| Prov96-204 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (3.7) | 1 (0.2) | NS |
| sonnei | 4 (28.6) | 1 (4.5) | 7 (11.9) | 9 (15.0) | 9 (11.0) | 8 (15.7) | 9 (34.6) | 4 (36.4) | 1 (12.5) | 18 (46.2) | 13 (29.5) | 7 (25.9) | 90 (20.3) | < 0.001 |
| dysenteriae† | 0 | 5 (22.7) | 11 (18.6) | 12 (20) | 15 (18.3) | 6 (11.8) | 6 (23.1) | 0 | 1 (12.5) | 3 (7.7) | 6 (13.6) | 7 (25.9) | 72 (16.3) | NS |
| 2 | 0 | 3 (13.6) | 8 (13.6) | 7 (11.7) | 9 (11.0) | 3 (5.9) | 1 (3.8) | 0 | 0 | 0 | 2 (4.5) | 0 | 33 (7.4) | NS |
| 3 | 0 | 2 (9.1) | 0 | 5 (8.3) | 5 (6.1) | 1 (2.0) | 1 (3.8) | 0 | 0 | 2 (5.1) | 3 (6.8) | 0 | 19 (4.3) | NS |
| 4 | 0 | 0 | 0 | 0 | 1 (1.2) | 2 (3.9) | 1 (3.8) | 0 | 0 | 0 | 0 | 2 (7.4) | 6 (1.3) | NS |
| 12 | 0 | 0 | 0 | 0 | 0 | 0 | 2 (7.7) | 0 | 0 | 1 (2.6) | 0 | 3 (11.1) | 6 (1.3) | 0.03 |
| boydii | 2 (14.3) | 2 (9.1) | 7 (11.9) | 4 (6.7) | 9 (11.0) | 4 (7.8) | 2 (7.7) | 2 (18.2) | 0 | 0 | 2 (4.5) | 0 | 34 (7.7) | NS |
| 1 | 1 (7.1) | 1 (4.5) | 2 (3.4) | 1 (1.7) | 2 (2.4) | 0 | 0 | 0 | 0 | 0 | 1 (2.3) | 0 | 8 (1.8) | NS |
| 2 | 0 | 0 | 0 | 2 (3.3) | 4 (4.9) | 0 | 1 (3.8) | 1 (9.1) | 0 | 0 | 0 | 0 | 8 (1.8) | NS |
| 4 | 0 | 0 | 3 (5.1) | 1 (1.7) | 0 | 1 (2.0) | 0 | 1 (9.1) | 0 | 0 | 0 | 0 | 6 (1.3) | NS |
| 18 | 1 (7.1) | 0 | 1 (1.7) | 0 | 1 (1.2) | 0 | 1 (3.8) | 0 | 0 | 0 | 0 | 0 | 4 (0.9) | NS |
| Undetermined | 0 (0) | 0 (0) | 0 (0) | 1 (1.7) | 0 (0) | 1 (2.0) | 0 (0) | 0 (0) | 1 (12.5) | 0 (0) | 1 (2.3) | 0 (0) | 4 (0.9) | NS |
Shigella flexneri serotypes proposed for inclusion, along with S. sonnei, in a quadrivalent broad-spectrum Shigella vaccine are shown in bold.
Only the four most frequent serotypes are shown.
An increase in the frequency of S. sonnei isolates relative to S. flexneri has been reported worldwide, in regions in which sanitation and clean water provision have been improved.14 Such interactions between these two serogroups were detected here, by analyzing the negative correlation between proportions and incidence, which was strong year after year (r = −0.8164; P = 0.001), whereas an erratic pattern was observed between years (Table 1). Five serotypes/subserotypes of S. flexneri accounted for 50.9% of all isolates: 6 (14.4% of the total), 1b (13.3%), 2a (9.0%), 3a (8.6%), and 4a (5.6%). Only minimal changes in serotype distribution were observed from year to year, for most of the (sub-)serotypes, and any significant variation detected was inconsistent (Table 1). Shigella dysenteriae serotype 1, which is a source of great concern as it has caused devastating epidemics of shigellosis in various developing countries, including CAR,15 was recovered only rarely in our laboratory (one organism in 2006).
No significant cross-reactions are known between serotypes in S. dysenteriae (15 serotypes) and S. boydii (20 serotypes), but major cross-reactions were observed for 14 of the 19 serotypes of S. flexneri, because of a degree of antigenic relatedness attributable to a common repeating tetrasaccharide unit.16 Thus, a multivalent vaccine including O antigens from S. flexneri 2a and 3a, in addition to direct protection against S. flexneri 2b and 3b, would provide cross-protection against S. flexneri 1a, 1b, 4a, 4b, 5a, 5b, 7b, X, and Y.17,18 Extrapolating these data to humans, a multivalent vaccine including S. sonnei (only one serotype described) and S. flexneri 2a, 3a, and 6 would have provided direct protection for 52.3% and cross-protection for 72.9% of these infections. This level is lower than that estimated for two multicenter studies at four sites in Africa and nine sites at Asia,5,19 in which a quadrivalent vaccine including the serotypes listed previously would have provided protection against at least 85% of the serotyped organisms. However, these cross-reactions remain theoretical and discrepancies exist between data for humans and animals, as highlighted by the appearance of cross-reactive type six antibodies in humans, but not in mice, after vaccination with S. flexneri 2a conjugate.20 Together with the considerable diversity of (sub-)serotypes in two of the three major serogroups (S. flexneri and S. dysenteriae) recovered in our study, this constitutes a real barrier to the development of a cheap, safe vaccine providing broad coverage against Shigella.
The ability of Shigella to acquire antimicrobial drug resistance rapidly is a major challenge in the control of infections with this bacterium. Overall, resistance rates to antimicrobials were low during the study period, for all classes other than conventional antimicrobials (chloramphenicol [62%], amoxicillin [64%], cotrimoxazole [92%], and tetracycline [98%]), confirming recommendations for first-line treatment based on C3G and fluoroquinolones (Table 2). However, although no resistance to ciprofloxacin was detected, we report the first case of ESBL-producing Shigella (S. flexneri) in sub-Saharan Africa, which is of major concern. Unlike S. sonnei, the S. flexneri, S. dysenteriae, and S. boydii serotypes were all strongly associated with high rates of MDR (Table 2). After adjustment for gender and serogroup, multivariate analysis highlighted a significant contribution of the antimicrobial drug resistance pattern of S. sonnei to the rates of MDR of Shigella isolates (OR = 0.02 95% CI [0.007–0.65]; P < 0.001), despite the lower prevalence of S. sonnei than that of S. flexneri (Table 3).
Table 2.
Percentage resistance to specific antibiotics in Shigella species in Bangui, Central African Republic, from 2002 to 2013
| Shigella flexneri (%) | Shigella sonnei (%) | Shigella dysenteriae (%) | Shigella boydii (%) | Unknown (%) | Total (%) | |
|---|---|---|---|---|---|---|
| N = 243 | N = 90 | N = 72 | N = 34 | N = 4 | N = 443 | |
| Amoxicillin | 72 | 19 | 85 | 68 | 100 | 64 |
| Co-amoxiclav | 6 | 2 | 1 | 3 | 0 | 4 |
| Ticarcillin | 71 | 19 | 85 | 65 | 100 | 61 |
| Cefoxitin | 0 | 0 | 0 | 0 | 0 | 0 |
| Cefotaxime | 0 | 0 | 0 | 0 | 0 | 0 |
| Amikacin | 0 | 0 | 0 | 0 | 0 | 0 |
| Gentamicin | 0 | 0 | 0 | 0 | 0 | 0 |
| Nalidixic acid | 0 | 0 | 1 | 0 | 0 | 0 |
| Ciprofloxacin | 0 | 0 | 0 | 0 | 0 | 0 |
| Chloramphenicol | 74 | 20 | 85 | 32 | 75 | 62 |
| Sulfonamides | 92 | 98 | 96 | 91 | 100 | 94 |
| Cotrimoxazole | 90 | 97 | 94 | 88 | 100 | 92 |
| Tetracycline | 98 | 99 | 99 | 97 | 100 | 98 |
Table 3.
Determinants of the high rates of multidrug resistance (defined by resistance to more than five of the 13 antibiotics tested) in Shigella spp. strains from Bangui, Central African Republic, 2002–2013
| Rate of multidrug resistance % (n) | Univariate analysis | Multivariate analysis | ||||
|---|---|---|---|---|---|---|
| Yes (N = 216) | No (N = 227) | OR (95% CI) | P | Adjusted OR (95% CI) | P | |
| Male | 48.6 (105) | 57.7 (131) | 0.72 (0.49–1.05) | 0.086 | 0.76 (0.50–1.14) | 0.189 |
| Q4 age* | 21.8 (47) | 23.3 (53) | 0.91 (0.58–1.43) | 0.689 | – | – |
| flexneri† | 62.5 (135) | 47.6 (108) | 1.84 (1.26–2.69) | 0.002 | 0.41 (0.04–3.85) | 0.436 |
| sonnei† | 6.9 (15) | 33.0 (75) | 0.15 (0.08–0.27) | < 0.0001 | 0.07 (0.01–0.66) | 0.019 |
| dysenteriae† | 25.6 (55) | 7.5 (17) | 4.22 (2.36–7.56) | < 0.0001 | 1.03 (0.10–10.05) | 0.978 |
| boydii† | 3.7 (8) | 11.5 (26) | 0.30 (0.13–0.67) | 0.003 | 0.10 (0.01–1.10) | 0.059 |
| Undefined† | 1.4 (3) | 0.5 (1) | 3.18 (0.33–31.03) | 0.317 | – | – |
Q4 age-defined extreme population quartile.
Serogroup.
The data reported here are particularly important, given the difficulty of carrying out such studies in countries with inadequate health-care systems. The high diversity of S. flexneri and S. dysenteriae (sub-)serotypes observed here may act as a major obstacle to the development of a vaccine. Resistance to front-line antimicrobials is low, but it will be important to continue monitoring antimicrobial drug susceptibility in Shigella isolates.
Acknowledgments:
We thank Isabelle Carle, Monique Lejay-Collin, Malika Gouali, and Corinne Ruckly for excellent technical assistance.
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