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Cholera in pregnancy: Clinical and immunological aspects

  • Ashraful I. Khan
    Affiliations
    Centre for Vaccine Sciences, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh
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  • Fahima Chowdhury
    Affiliations
    Centre for Vaccine Sciences, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh
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  • Daniel T. Leung
    Affiliations
    Centre for Vaccine Sciences, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh

    Division of Infectious Diseases, University of Utah, Salt Lake City, UT, USA
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  • Regina C. Larocque
    Affiliations
    Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA

    Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Jason B. Harris
    Affiliations
    Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA

    Department of Medicine, Harvard Medical School, Boston, MA, USA
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  • Edward T. Ryan
    Affiliations
    Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA

    Department of Medicine, Harvard Medical School, Boston, MA, USA

    Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA
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  • Stephen B. Calderwood
    Affiliations
    Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA

    Department of Medicine, Harvard Medical School, Boston, MA, USA

    Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA
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  • Firdausi Qadri
    Correspondence
    Corresponding author. Centre for Vaccine Sciences, icddr,b, 68 Shahid Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh. Tel.: +880 2 9827001 10; fax: +880 2 8823116/2 8826050x2431.
    Affiliations
    Centre for Vaccine Sciences, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh
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Open AccessPublished:August 14, 2015DOI:https://doi.org/10.1016/j.ijid.2015.08.006

      Highlights

      • Pregnant and non-pregnant women with cholera had similar clinical severity and comparable immune responses.
      • We did not observe adverse foetal outcomes in 21 days of follow up after cholera in this cohort presenting at a median of 14 weeks of gestation.
      • These data support further study of the use of oral killed cholera vaccine in pregnant women at risk for cholera in cholera endemic areas.
      • Our study was limited to individuals with severe cholera, and immunological responses in mild or asymptomatic cases were not evaluated in the cohort.

      Summary

      Background

      The objective of this study was to examine the clinical and immunological features of cholera in pregnancy.

      Methods

      Women of reproductive age presenting to the icddr,b Dhaka hospital with cholera, and enrolled as part of a larger cohort study, were tested for pregnancy on admission. We compared initial clinical features and immune responses of pregnant patients with non-pregnant female patients at days 2, 7 and 21 after infection.

      Results

      Among reproductive age women enrolled between January 2001 and May 2006, 9.7% (14/144) were pregnant. The duration of diarrhoea prior to admission tended to be higher in pregnant compared to non-pregnant patients (p=0.08), but other clinical characteristics did not differ. Antibody responses to cholera toxin B subunit (CtxB), toxin-coregulated pilus A (TcpA), Vibrio cholerae lipopolysaccharide (LPS), and serum vibriocidal antibody responses, were comparable between pregnant and non-pregnant patients. There were no deaths among the pregnant cases or non-pregnant controls, and no adverse foetal outcomes, including stillbirths, during 21 days of follow up of pregnant cases.

      Conclusions

      To our knowledge, this is the first report of immune responses in pregnant women with cholera. We found that pregnant woman early in pregnancy has comparable clinical illness and subsequent immune responses compared to non-pregnant women. These findings suggest that the evaluation of safety and immunogenicity of oral cholera vaccines in pregnancy should be an area of future investigations.

      Keywords

      1. Introduction

      Cholera is a life threatening diarrheal disease caused predominantly by infection with Vibrio cholerae O1. Though cholera is rare in developed countries, it is prevalent in many areas of South and Southeast Asia and in Africa and may also cause major outbreaks worldwide.
      • WHO
      Cholera vaccines: WHO position paper.
      Bangladesh is a country in South Asia where cholera is endemic and is consistently present throughout the year in high risk areas.
      • Ali M.
      • Emch M.
      • Donnay J.-P.
      • Yunus M.
      • Sack R.
      The spatial epidemiology of cholera in an endemic area of Bangladesh.
      Cholera toxin (CT), the primary toxin produced by V. cholerae O1 and O139, causes the hypersecretion of electrolytes and water, sometimes with fatal results. The lipopolysaccharide of V. cholerae is an important determinant of protection, and is the primary antigen found in the most recent formulations of the oral cholera vaccine (OCV).
      Pregnancy is an immuno-altered state where both humoral and cellular immunity are affected.
      • Armenti V.T.
      • Moritz M.J.
      • Cardonick E.H.
      • Davison J.M.
      Immunosuppression in pregnancy: choices for infant and maternal health.
      • Mor G.
      • Cardenas I.
      The immune system in pregnancy: a unique complexity.
      Several pregnancy outcomes, including preeclampsia, poor foetal growth, and preterm birth, have been linked to abnormalities in immune responses during pregnancy.
      • Murtha A.P.
      • Sinclair T.
      • Hauser E.R.
      • Swamy G.K.
      • Herbert W.N.
      • Heine R.P.
      Maternal serum cytokines in preterm premature rupture of membranes.
      • Borzychowski A.M.
      • Sargent I.L.
      • Redman C.W.
      Inflammation and pre-eclampsia.
      • Conrad K.P.
      • Miles T.M.
      • Benyo D.F.
      Circulating levels of immunoreactive cytokines in women with preeclampsia.
      Pregnancy has also been associated with decreased inflammatory responses and increased anti-inflammatory responses to immune challenges in humans as well as in animal models.
      • Fofie A.E.
      • Fewell J.E.
      • Moore S.L.
      Pregnancy influences the plasma cytokine response to intraperitoneal administration of bacterial endotoxin in rats.
      • Aguilar-Valles A.
      • Poole S.
      • Mistry Y.
      • Williams S.
      • Luheshi G.N.
      Attenuated fever in rats during late pregnancy is linked to suppressed interleukin-6 production after localized inflammation with turpentine.
      In some cases, pregnant women are more susceptible to certain infections, and when infected, may experience a higher severity of illness. For example, pregnant women infected with influenza virus are at increased risk for serious complications when compared to other groups,
      • Jamieson D.J.
      • Honein M.A.
      • Rasmussen S.A.
      • Williams J.L.
      • Swerdlow D.L.
      • Biggerstaff M.S.
      • et al.
      H1N1 2009 influenza virus infection during pregnancy in the USA.
      though a recent study on influenza virus vaccine during pregnancy showed that pregnancy did not significantly alter antibody responses.
      • Christian L.M.
      • Porter K.
      • Karlsson E.
      • Schultz-Cherry S.
      • Iams J.D.
      Serum proinflammatory cytokine responses to influenza virus vaccine among women during pregnancy versus non-pregnancy.
      Women living in areas endemic for cholera are at risk of acquiring the disease during pregnancy, and studies from South Asia, Africa, and Haiti have demonstrated that cholera during pregnancy may increase the risk of poor outcomes.
      • Ciglenecki I.
      • Bichet M.
      • Tena J.
      • Mondesir E.
      • Bastard M.
      • Tran N.-T.
      • et al.
      Cholera in pregnancy: outcomes from a specialized cholera treatment unit for pregnant women in Leogane, Haiti.
      However, there is a lack of data on the immunological responses to cholera during pregnancy to determine if vaccination might play a role in prevention. Thus, the objective of this study was to examine the clinical characteristics and immunological responses of pregnant women following severe cholera.

      2. Materials and Methods

      2.1 Study population and patient enrolment

      The Cholera Immune Response Study (CIRS) was a prospective, observational study, undertaken as a collaboration between the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) and Massachusetts General Hospital in Boston. The icddr,b in Dhaka, Bangladesh cares for approximately 120,000 patients with diarrheal diseases each year. Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1) were eligible for inclusion in this study if their stool cultures were subsequently positive for V. cholerae, and if they were without significant co-morbid conditions; patients were enrolled prospectively in the study between 2001 and 2006.
      • Harris J.B.
      • Khan A.I.
      • LaRocque R.C.
      • Dorer D.J.
      • Chowdhury F.
      • Faruque A.S.
      • et al.
      Blood group, immunity, and risk of infection with Vibrio cholerae in an area of endemicity.
      • Saha D.
      • LaRocque R.C.
      • Khan A.I.
      • Harris J.B.
      • Begum Y.A.
      • Akramuzzaman S.M.
      • et al.
      Incomplete correlation of serum vibriocidal antibody titer with protection from Vibrio cholerae infection in urban Bangladesh.
      Stool cultures for V. cholerae were done on taurocholate-tellurite-gelatin agar (TTGA). After overnight incubation of plates, serological confirmation of suspected V. cholerae colonies was carried out by slide agglutination.
      • Rahman M.
      • Sack D.
      • Mahmood S.
      • Hossain A.
      Rapid diagnosis of cholera by coagglutination test using 4-h fecal enrichment cultures.
      Patients were enrolled on day 2 of admission (study day 2) if a stool culture was positive for V. cholerae O1 or O139. Information regarding clinical features, demographics, and history of diarrhoea were collected from patients at enrolment. Samples of venous blood were collected, for determining antibody titers, from patients on study day 2 and again at follow-up visits on study days 7 and 21. Informed written consent for participation in this research was obtained from participants or their guardians. This study was reviewed and approved by the Ethical and Research Review Committees of the icddr,b and the Institutional Review Board of the Massachusetts General Hospital.

      2.2 Pregnant women and case control comparison

      All women of reproductive age (15-49) enrolled in the CIRS study were screened for pregnancy by urine strip test (hCG One Step Pregnancy Test Strip, TUV product service, USA) on enrolment. A total of 14 women had a positive pregnancy test. We also selected all non-pregnant cases as controls from the same age cohort.

      2.3 Treatment of patients

      Patients enrolled for the study received the normal standard of care provided at the icddr,b for cholera. Dehydration was corrected either by infusing intravenous cholera saline or by oral rehydration solution depending on the severity of the dehydration and clinical condition of the patient. A short course of oral antibiotics was given. Non pregnant adult females with stool culture positive for V. cholerae received 300 mg of doxycycline in a single dose, whereas pregnant women with cholera received erythromycin (500 mg every six hours) for three days.

      2.4 Immunological assessment

      We measured vibriocidal antibodies in patient plasma using V. cholerae O1 El Tor Ogawa (strain X25049) or Inaba (strain 19479) or V. cholerae O139 (strain 4260B) as target bacteria, and adding guinea pig complement.
      • Qadri F.
      • Mohi G.
      • Hossain J.
      • Azim T.
      • Khan A.
      • Salam M.
      • et al.
      Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.
      Antibody responses to recombinant cholera toxin B subunit (CtxB), toxin-coregulated pilus A subunit (TcpA), and lipopolysaccharide (LPS) were determined using a kinetic enzyme-linked immunosorbent assay (ELISA), as previously described.
      • Asaduzzaman M.
      • Ryan E.T.
      • John M.
      • Hang L.
      • Khan A.I.
      • Faruque A.
      • et al.
      The major subunit of the toxin-coregulated pilus TcpA induces mucosal and systemic immunoglobulin A immune responses in patients with cholera caused by Vibrio cholerae O1 and O139.
      In all cases, the vibriocidal and LPS responses were measured to the same serotype, Ogawa or Inaba of V. cholerae O1 and V. cholerae O139 as was present in the patient.

      2.5 Data analyses

      Data analyses were performed using SPSS for Windows (version 12; SPSS Inc., Chicago, IL, USA) and Epi Info (version 6.0; USD, Stone Mountain, GA, USA). For non-normally distributed data, comparisons were carried out as median (25th–75th percentile) using the Mann–Whitney U-test. The significance of differences in proportions was evaluated by the Chi-square test, and Fisher's exact test was applied when appropriate. A p-value of <0.05 was considered statistically significant.

      3. Results

      A total of 399 patients with cholera were enrolled in the study between January 2001 and May 2006 and the data obtained prospectively were analysed in a retrospective fashion. Overall, 144 cholera patients were reproductive age (15-49 years) women and of those, 9.7% (14/144) had a positive pregnancy test. Average duration of pregnancy was 14 weeks at enrolment (minimum 6 weeks, maximum 24 weeks, mean ±SD = 14±5.6 weeks) (data not shown). The clinical and microbiological features of the pregnant and non-pregnant cholera cases are presented in Table 1.
      Table 1Clinical and microbiological features of pregnant and all non-pregnant study participants
      CharacteristicsPregnant (n=14)Non-pregnant (n=130)p-value
      Demographics
      Median age (yr), (25th, 75th percentile)24.5 (21 - 29)27 (21 - 36)0.14
      Blood group, n (%)
       O4 (28.6)58 (44.6)
       A2 (14.3)36 (27.7)
       B6 (42.9)31 (23.9)
       AB2 (14.3)5 (3.9)0.09
      Clinical
      Dehydration
       Some1 (7.1)9 (6.9)
       Severe13 (92.9)121 (93.18)1.00
      No. of stools (> 20 in last 24hrs)10 (71.4)111 (85.48)0.24
      Fever in the last week2 (14.3)27 (20.8)0.74
      Vomiting in the last 24hrs
       No or <10 times5 (35.7)65 (50.0)
       ≥ 10 times9 (64.3)65 (50.0)0.40
      Abdominal pain at presentation11 (78.6)93 (71.5)0.76
      Helminth present2 (14.3)16 (12.3)0.69
      Received IV fluid13 (92.9)126 (96.9)0.41
      Duration of diarrhoea, hr
       Median (IQR)100 (9 - 105)12 (7 - 23)0.08
      Duration of stay, hr
       Median (IQR)102 (41 - 104)103 (100 - 105)0.25
      Microbiological, n (%)
       Dark field positive stool
      V. cholerae O1, Ogawa3 (21.4)41 (31.5)0.55
      V. cholerae O1, Inaba7 (50.0)66 (50.8)1.00
      V. cholerae O1394 (28.6)23 (17.7)0.30
      The duration of diarrhoea prior to admission trended toward higher (p=0.08) in the pregnant cohort compared to the non-pregnant control cohort of patients with cholera. However, other clinical parameters, including severity of dehydration and volume of intravenous fluids required, did not differ between the pregnant and non-pregnant cholera cases (Table 1). There were no deaths among the pregnant cases or non-pregnant controls, and no adverse foetal outcomes including spontaneous abortions in the 21 days following cholera in the pregnant women.
      We assessed the association between the baseline (Day 2) and convalescent stage (Day 7 and 21) responses in a number of immunologic markers and pregnancy status of cholera patients. Serum vibriocidal titers (Table 2), as well as antibody responses to LPS, CtxB, and TcpA (Table 3, Table 4, Table 5), showed no statistically significant differences between pregnant and non-pregnant patients overall. However, when we compared responses separately for V. cholerae O1 and O139 infected patients, serum vibriocidal responses and as well as antibody response to LPS and CtxB were higher in non-pregnant women compared to pregnant women infected with V. cholerae O139 (Table 2, Table 3, Table 4).
      Table 2Vibriocidal antibody responses of study participants by pregnancy status
      Infecting strainAntibodyPregnant (n=14)Non-pregnant (n=130)p-value
      Vibriocidal

      (
      Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1); Antibody titers, from patients at baseline on study day 2 and again at follow-up visits on study days 7 and 21.
      Study Days)
      nMedian (IQR)nMedian (IQR)
      OverallDay 21480 (10 - 640)12880 (20 - 160)0.84
      Day 7142560 (1280 - 8960)1225120 (2560 - 10240)0.32
      Day 21112560 (640 - 5120)1202560 (1280 - 5120)0.66
      V.cholerae O1Day 210480 (50 - 640)10580 (40 - 320)0.19
      Day 7103840 (2560,17920)995120 (2560 - 10240)0.97
      Day 2195120 (1280,5120)975120 (1280 - 5120)0.95
      V. cholerae O139Day 248 (5 -,28)2320 (10 - 80)0.27
      Day 74640 (520 - 800)231280 (640 - 2560)0.10
      Day 212163 (84 - 241)23640 (640 - 1280)0.05
      * Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1); Antibody titers, from patients at baseline on study day 2 and again at follow-up visits on study days 7 and 21.
      Table 3Responses to LPS of study participants by pregnancy status
      Infecting strainAntibody

      (
      Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1); Antibody titers, from patients at baseline on study day 2 and again at follow-up visits on study days 7 and 21.
      Study Days)
      Pregnant (n=14)Non-pregnant (n=130)p-value
      nMedian (IQR)nMedian (IQR)
      OverallLPS IgA
      Day 21422 (18 - 31)13022 (12 - 39)0.95
      Day 714103 (32 - 250)124151 (73 - 268)0.26
      Day 211181(30 - 177)12097 (49 - 184)0.49
      LPS IgG
      Day 21472 (47 - 122)13079 (55 - 106)0.87
      Day 714180 (98 - 248)124227 (164 - 292)0.12
      Day 2111258 (106 - 283)120238 (150 - 313)0.30
      V.cholerae O1LPS IgA
      Day 21024 (19 - 31)10721 (11 - 37)0.49
      Day 710169 (87 - 308)101162 (74 - 293)0.97
      Day 219106 (30 - 184)9789 (43 - 200)0.80
      LPS IgG
      Day 21099 (60 - 138)10775 (55 - 103)0.30
      Day 710224 (158 - 259)101226 (163 - 297)0.73
      Day 219268 (123 - 298)97230 (146 - 318)0.91
      V. cholerae O139LPS IgA
      Day 2421 (14 - 26)2330 (14 - 42.67)0.25
      Day 7438 (21 - 54.17)23102 (64 - 170)0.02
      Day 21247 (38 - 55.33)23117 (68 - 158)0.09
      LPS IgG
      Day 2453 (37 - 64)2390 (72 - 111)0.02
      Day 74107 (81 - 130)23228 (195 - 277)0.01
      Day 21283 (75 - 91)23246 (219 - 300)0.03
      * Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1); Antibody titers, from patients at baseline on study day 2 and again at follow-up visits on study days 7 and 21.
      Table 4Responses to CtxB of study participants by pregnancy status
      Infecting strainAntibody

      (
      Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1); Antibody titers, from patients at baseline on study day 2 and again at follow-up visits on study days 7 and 21.
      Study Days)
      Pregnant (n=14)Non-pregnant (n=130)p-value
      nMedian (IQR)nMedian (IQR)
      OverallCtxB IgA
      Day 21419 (16 - 38)13022 (12 - 44)0.75
      Day 71492 (58 - 160)124123 (65 - 204)0.49
      Day 211156 (48 - 115)12071 (41 - 125)0.89
      CtxB IgG
      Day 214109 (75 - 131)13098 (61 - 157)0.87
      Day 714288 (213 - 352)124260 (194 - 425)0.98
      Day 2111369 (252 - 401)120285 (213 - 399)0.40
      V.cholerae O1CtxB IgA
      Day 21020 (16 - 60)10722 (12 - 41)0.75
      Day 71092 (64 - 156)101111 (57 - 175)0.96
      Day 21968 (48 - 135)9767 (36 - 100)0.44
      CtxB IgG
      Day 210117 (85 - 131)10799 (63 - 160)0.70
      Day 710288 (266 - 347)101260 (191 - 411)0.52
      Day 219378 (281 - 411)97286 (202 - 410)0.13
      V. cholerae O139CtxB IgA
      Day 2419 (16 - 20)2325 (15 - 49)0.19
      Day 7497 (52 - 151)23191 (129 - 304)0.08
      Day 21250 (49 - 51)23107 (74 - 176)0.04
      CtxB IgG
      Day 2492 (63 - 119)2375 (53 - 132)1.00
      Day 74231 (124 - 327)23260 (219 - 430)0.25
      Day 212180 (151 - 209)23282 (234 - 385)0.11
      * Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1); Antibody titers, from patients at baseline on study day 2 and again at follow-up visits on study days 7 and 21.
      Table 5Responses to TcpA of study participants by pregnancy status
      Infecting strainAntibody

      (Study Days)
      Pregnant (n=14)Non-pregnant (n=130)p-value
      nMedian (IQR)nMedian (IQR)
      OverallTcpA IgA
      Day 295 (2 - 5)796 (3 - 11)0.08
      Day 7913 (7 - 33)7722 (7 - 46)0.91
      Day 21912 (9 - 37)7611 (6 - 26)0.26
      TcpA IgG
      Day 2730 (20 - 40)5840 (24 - 65)0.21
      Day 7766 (65 - 142)5664 (35 - 128)0.41
      Day 21790 (58 - 130)5577 (44 - 120)0.47
      V.cholerae O1TcpA IgA
      Day 273 (2 - 5)686 (3 - 11)0.07
      Day 7713 (6 - 23)6623 (6 - 46)0.62
      Day 21712 (10 - 35)6513 (6 - 27)0.44
      TcpA IgG
      Day 2626 (20 - 35)5742 (24 - 65)0.15
      Day 7666 (64 - 110)5556 (35 - 123)0.65
      Day 216100 (51 - 131)5476 (40 - 122)0.46
      V. cholerae O139TcpA IgA
      Day 225 (4.6 - 4.9)115 (4 - 5)1.00
      Day 72104 (57 - 152)1114 (7 - 36)0.43
      Day 21247 (28 - 66)1110 (8 - 11)0.43
      Tcp IgG
      Day 2143 (43 - 43)126 (26 - 26)0.32
      Day 71160 (160 - 160)1127 (127 - 127)0.32
      Day 21190 (90 - 90)1101 (101 - 101)0.32
      * Patients presenting to the icddr,b Dhaka hospital with acute watery diarrhoea (study day 1); Antibody titers, from patients at baseline on study day 2 and again at follow-up visits on study days 7 and 21.

      4. Discussion

      This is the first report to our knowledge of immune responses following cholera in pregnant women. We found that overall, most of the clinical and immunological characteristics were comparable between pregnant and non-pregnant women presenting to the icddr,b hospital with severe cholera. When analysed separately for patients infected with V .cholerae O1 versus O139, the vibriocidal antibody responses following O1 infection were more robust in general than those seen following O139 infection, as we have seen earlier.
      • Qadri F.
      • Mohi G.
      • Hossain J.
      • Azim T.
      • Khan A.
      • Salam M.
      • et al.
      Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.
      Serum vibriocidal responses as well as antibody responses to LPS, CtxB and TcpA were similar between pregnant and non-pregnant women infected with V. cholerae O1. Pregnant women infected with V. cholerae O139 had lower serum vibriocidal responses and antibody responses to LPS and CtxB compared to non-pregnant women infected with this serogroup. This study also found no deaths among the pregnant cases or non-pregnant controls, and no adverse foetal outcomes during 21 days of follow up of the pregnant cases. Of note, our follow up period was limited to 21 days after infection, and the infected pregnant women were in the first or second trimester.
      In a study from Haiti, a previously non-endemic area, analysis of outcome data from a large cohort of pregnant women with cholera revealed that the main risk factor for foetal death was severity of dehydration.
      • Ciglenecki I.
      • Bichet M.
      • Tena J.
      • Mondesir E.
      • Bastard M.
      • Tran N.-T.
      • et al.
      Cholera in pregnancy: outcomes from a specialized cholera treatment unit for pregnant women in Leogane, Haiti.
      Although our study did not follow the outcome of pregnancy for more than the 21 day follow up period after the episode of cholera, we did not find any difference in the severity of dehydration between pregnant and non-pregnant women presenting in the first or second trimester with severe cholera. At icddr,b severe dehydration is immediately corrected with cholera saline following presentation to the facility. The management of individuals with cholera involves fluid resuscitation based on the level of dehydration and then after adequate hydration, maintenance of ongoing fluid losses.
      • Chowdhury F.
      • Khan A.I.
      • Faruque A.S.G.
      • Ryan E.T.
      Severe, acute watery diarrhea in an adult.
      Cholera in the third trimester has previously been significantly associated with an increased severity of diarrhoeal disease, as indicated by severe dehydration on admission to hospital, as compared with non-pregnant controls.
      • Hirschhorn N.
      • Chowdhury A.K.
      • Lindenbaum J.
      Cholera in pregnant women.
      Several studies also suggest an association between foetal loss and the degree of dehydration and hypovolemia.
      • Hirschhorn N.
      • Chowdhury A.K.
      • Lindenbaum J.
      Cholera in pregnant women.
      • Saona P.A.J.
      • Figueroa M.
      • Maradiegue E.
      Cholera in pregnant women at the Hospital Nacional Cayetano Heredia, Lima Perú.
      • Diop S.
      • Manga N.
      • Dia N.
      • Gaye S.
      • Ndour C.
      • Seydi M.
      • et al.
      [Cholera and pregnancy: epidemiological, clinical, and evolutionary aspects].
      Independent of the dehydration status, severe vomiting was also found to be another risk factor for foetal death.
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      • Bichet M.
      • Tena J.
      • Mondesir E.
      • Bastard M.
      • Tran N.-T.
      • et al.
      Cholera in pregnancy: outcomes from a specialized cholera treatment unit for pregnant women in Leogane, Haiti.
      Changes in electrolytes in the amniotic fluid of pregnant women suffering from cholera have been described in the literature,
      • Ogunbode O.
      • Onifade A.
      Amniotic fluid electrolytes, urea and creatinine in normal pregnancy and cholera during pregnancy.
      but it remains unclear how these affect the foetus. In our study, it is notable that vomiting was not significantly different between pregnant patients in the first and second trimester and non-pregnant cholera patients.
      The serum vibriocidal antibody assay has been used to measure the antibacterial antibody response in cholera patients and elevated vibriocidal antibody levels have been shown to correlate with protection against cholera.
      • Qadri F.
      • Mohi G.
      • Hossain J.
      • Azim T.
      • Khan A.
      • Salam M.
      • et al.
      Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.
      • Glass R.I.
      • Svennerholm A.-M.
      • Khan M.
      • Huda S.
      • Huq M.I.
      • Holmgren J.
      Seroepidemiological studies of El Tor cholera in Bangladesh: association of serum antibody levels with protection.
      • Harris J.B.
      • LaRocque R.C.
      • Chowdhury F.
      • Khan A.I.
      • Logvinenko T.
      • Faruque A.
      • et al.
      Susceptibility to Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh.
      This has led to the use of serum vibriocidal antibody responses as a surrogate marker for cholera vaccine efficacy. In our study, serum vibriocidal responses did not differ between pregnant and non-pregnant cholera patients infected with V. cholerae O1, the major currently circulating serogroup.
      LPS is an important antigen for enteric pathogens and responses to LPS appear to be involved in protection against cholera.
      • Leung D.T.
      • Uddin T.
      • Xu P.
      • Aktar A.
      • Johnson R.A.
      • Rahman M.A.
      • et al.
      Immune responses to the O-specific polysaccharide antigen in children who received a killed oral cholera vaccine compared to responses following natural cholera infection in Bangladesh.
      For example, V. cholerae O1 LPS has been shown to induce protective immune responses in humans and animals
      • Apter F.
      • Michetti P.
      • Winner L.d.
      • Mack J.
      • Mekalanos J.
      • Neutra M.
      Analysis of the roles of antilipopolysaccharide and anti-cholera toxin immunoglobulin A (IgA) antibodies in protection against Vibrio cholerae and cholera toxin by use of monoclonal IgA antibodies in vivo.
      • Qadri F.
      • Ahmed F.
      • Karim M.M.
      • Wenneras C.
      • Begum Y.A.
      • Salam M.A.
      • et al.
      Lipopolysaccharide- and cholera toxin-specific subclass distribution of B-cell responses in cholera.
      and thus its use as a protective immunogen for cholera vaccine development has been widely accepted.
      • Winner L.
      • Mack J.
      • Weltzin R.
      • Mekalanos J.
      • Kraehenbuhl J.
      • Neutra M.
      New model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin A from hybridoma tumors protects against Vibrio cholerae infection.
      • Svennerholm A.M.
      • Holmgren J.
      Synergistic protective effect in rabbits of immunization with Vibrio cholerae lipopolysaccharide and toxin/toxoid.
      We did not find any statistically significant differences in LPS antibody responses between pregnant and non-pregnant cholera patients infected with V. cholerae O1. In cholera-endemic countries like Bangladesh, vaccinating the entire population may not be possible. However vaccination can be targeted in high-risk areas and to select high-risk populations that are especially vulnerable to severe disease; these targeted groups might include pregnant women if the vaccine is found to be safe in pregnancy. One recent study in Zanzibar found no statistically significant evidence of a harmful effect of gestational exposure to oral cholera vaccine.
      • Hashim R.
      • Khatib A.M.
      • Enwere G.
      • Park J.K.
      • Reyburn R.
      • Ali M.
      • et al.
      Safety of the recombinant cholera toxin B subunit, killed whole-cell (rBS-WC) oral cholera vaccine in pregnancy.
      Thus the evaluation of oral cholera vaccines and subsequent use in pregnant women should be considered an important priority.
      The main limitation of this study is the fact that the cohort consisted of only patients with severe cholera requiring hospitalization. Since many cases of infection with V. cholerae O1 do not result in severe infection, the association between pregnancy and the full spectrum of disease severity cannot be gauged in this study. Another limitation of this study is that we had a small sample size (14 pregnant women with cholera) and only followed pregnancy outcomes until 21 days after infection and we did not gather data on later foetal outcomes. Also, we did not assess mucosal immune responses using the antibody-secreting cell or antibody-in-lymphocyte supernatant assay for all the patients in this study. As most of the pregnant women were in the initial stage of gestation (average duration of pregnancy was 14 weeks), this may explain why the women had similar clinical severity unlike other studies later in pregnancy. The small sample size of pregnant women assessed for clinical severity, as well as the inclusion of only women with severe cholera, suggest that we did not have sufficient power for a full accounting of the severity of disease in pregnancy. Despite these limitations, to our knowledge, our study represents a unique analysis of immune responses to cholera during pregnancy.
      In conclusion, we have shown that pregnant women with severe cholera due to V. cholerae O1 respond immunologically in a similar manner to non-pregnant women despite the immune alterations associated with pregnancy. Our data underscore the fact that pregnant women with cholera are able to mount normal vibriocidal and other anti-V. cholerae immune responses. These findings suggest that the evaluation of safety and immunogenicity of oral cholera vaccines in pregnancy should be an area of future investigations so that the use of oral killed cholera vaccine in pregnant women at risk of cholera can be considered.

      Acknowledgements

      This study was supported by the following grants: U01 AI058935; AI106878 and K08 AI100923. Additionally the study was also supported by core grants to the icddr,b. The icddr,b is thankful to the Governments of Australia, Bangladesh, Canada, Sweden and the UK for providing core/unrestricted support. We wish to thank the study participants and the dedicated clinical, field and laboratory and data management staff of the Cholera Immune Response study.
      Conflicts of Interest: There are no conflicts of interest by authors.

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