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2018;2(5). https://doi.org/10.24087/IAM.2018.2.5.448. Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan.
May, 2018

2018; Vol2; Issue5

http://iamresearcher.online

Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan Lameck Nyangena Ontweka1, Margaret W. Muturi1, Francisco Luquero2 1

Department of Medical Laboratory Sciences, Kenyatta University, Nairobi, Kenya 2 Department of International Health, John Hopkins University, Baltimore, Mary Land, USA

Corresponding Author: Lameck Nyangena Ontweka [email protected]

ABSTRACT Cholera incidences are escalating in previously non-endemic zones. The situation is exacerbated by defective epidemiological surveillance systems as well as inadequate drug resistance profiling of Vibrio cholerae serotypes. This study assessed prevalence of V. cholerae serotypes and their antimicrobial susceptibility profiles in Juba, South Sudan. A cross-sectional laboratory-based experimental study was conducted on 109 consenting participants. Stool samples were collected and tested for V. cholerae using both conventional stool culture and serological approaches to determine the serotypes present. The latter was undertaken using both polyvalent and monovalent antisera. In addition, antimicrobial susceptibility profiling was conducted on study samples to determine drug sensitivity pattern. Demographic information showed that 70.6% (n=24) of all V. cholerae infections were among males aged (26-30 yrs; 32.4%). Of the total 109 stool samples only 31.2 % (n=34) were positive for V. cholerae; Inaba serotype. Antimicrobial susceptibility pattern revealed that V. cholerae was most sensitive to ceftriaxone, ciprofloxacin, norfloxacin and tetracycline antibiotics. Conversely, V. cholerae demonstrated highest resistance against nalidixic acid. Findings generated from the study inform on optimal selection of antibiotics for management of cholera while also providing up to date epidemiological information on V. cholerae serotypes present in Juba, Southern Sudan. Keywords: Antimicrobial susceptibility, prevalence, Cholera, Serotypes, Vibrio cholerae, Juba

1. INTRODUCTION Cholera is an acute diarrhea disease caused by bacilli Vibrio cholera sero group O1 or O139. The World Health Organization (WHO) estimates that 3–5 million annual cases of cholera occur worldwide, resulting in 100,000– 120,000 deaths [1]. Since its introduction to Africa during the seventh cholera pandemic in 1970, Vibrio cholerae caused regular vast epidemics across the continent with cumulative 3,762,902 case-notifications to WHO by 2013 [1]. In 2013 alone, 22 African countries reported 56,329 cholera cases. However, these numbers are considered to be substantially underestimated due to poorly functioning national epidemiological and laboratory surveillance systems, which are not able to detect the majority of mild disease presentations. Furthermore, systematic underreporting is common to avoid economic and political damage [2].

To Cite This Article: Lameck Nyangena Ontweka, Margaret W. Muturi, Francisco Luquero. Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan. International Annals of Medicine. 2018;2(5). https://doi.org/10.24087/IAM.2018.2.5.448

International Annals of Medicine Vol1;2(5);2018 Cholera remains a potential threat to South Sudan and it is likely to recur with such low indicators of social development. It remains a challenge in states where access to safe drinking water and adequate sanitation cannot be guaranteed [3]. Between April and October 2006, W.H.O reported 8200 cases including more than 230 deaths from cholera in northern Sudan. The situation is further aggravated by significant population movements including the increasing number of returning Internally Displaced Persons (IDPs), both from the North and the South Sudan. South Sudan represents a cholera endemic zone with 48,035 cholera outbreaks annually [4]. The transitional areas are also at risk of epidemics [3], since South Sudan has a fragile health system, with little capacity and a chronic lack of human resources. Cholera infection is associated with different clinical strains of V. cholerae. Approximately 200 sero-groups have been identified however only two sero-groups namely O1 and O139 have pathogenic strains [5]. Additionally, within V. cholerae sero-groups, there are two main serotypes that show variation in prevalence over time [6]. These biotypes are further grouped into 3 serotypes namely Ogawa, Inaba and Hikojima. In Africa, O1 sero-group shows highest prevalence in V. cholerae isolated cases [7]. It has been noted that majority of clinically isolated V. cholerae O1 strains are resistant to routinely administered antibiotics (nalidixic acid, trimethoprim/sulfamethoxazole and tetracycline). Information on cholera studies in Southern Sudan is also scanty. The prevalence and antimicrobial susceptibility profiles of Vibrio cholerae serotypes in Juba, South Sudan has not been determined. This study determined the prevalence of cholera serotypes and their antimicrobial resistance profiles in clinical isolates in Juba, South Sudan. The study recorded cholera prevalence of 31.2% which is higher than the one reported by WHO (5-10%) and high resistance to nalidixic acid in the region. Monitoring of epidemiological trends of V. cholerae strains will be important in the control of Cholera. The right treatment regimens should be recommended for the people living in this region [7].

2. METHODS Sampling Technique The study enrolled patients exhibiting clinical signs and symptoms of cholera at CTCs and JTH locations only. Consecutive sampling method was employed for all the patients that met the inclusion criteria. Those

who consented into the study were selected. Calculated sample size was based on World Health Organization (WHO) reports indicating current cholera prevalence rate of 5-10% and the Fishers (1998) [8] formulae giving a total of 109 samples. The study was approved by the John Hopkins Bloomberg School of Public Health Institutional Review Board and South Sudan Ethical Review Committee (IRB 00006548). Sample Collection and Preparation Labeled sterile buckets were placed under individual beds to collect stool samples from patients. A loopful of stool sample was aseptically picked and introduced into labeled sterile containers. Contents were immediately transported to the Public health laboratory for culture. Bacterial preserved in 0.9% w/v normal saline solution for viability under ambient temperatures. Stool Culture and Serological Test for Vibrio cholerae Serotypes A loopful of preserved stool samples was incubated for 8 hrs in alkaline peptone water (APW) at 37oC and later cultured into; thiosulfate citrate bile salt (TCBS), MacConkey and blood agars. All culture plates were incubated aerobically overnight at 37oC. Culture, microscopy and biochemical tests were performed for further V. Cholerae identification. Slide agglutination was performed to confirm the serotypes of V. cholerae isolates. Polyvalent V. cholerae O1 and V. cholerae O139 antisera and monovalent Ogawa and Inaba antisera were used. Antimicrobial Susceptibility Testing The Kirby-Bauer disc diffusion method was used for V. cholerae antimicrobial susceptibility testing [9]. Mueller Hinton agar medium was employed to test for antibiotic sensitivity patterns with Escherichia coli ATCC 25922 being used as the control standard organism. The diameters of zones of inhibition were measured to the nearest millimeter by use of a Vanier caliper and confirmation performed by digital zone inhibition reader (Fisher Scientific™, Pittsburgh, USA). Thereafter susceptibility profile against specific antibiotics was interpreted as susceptible (S), intermediate (I), resistant (R) and reference performed against Clinical Laboratory Standards Institute guidelines table [10]. Statistical Analysis The data was expressed as means ± standard error of mean (SEM). Analysis was conducted using statistical

To Cite This Article: Lameck Nyangena Ontweka, Margaret W. Muturi, Francisco Luquero. Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan. International Annals of Medicine. 2018;2(5). https://doi.org/10.24087/IAM.2018.2.5.448

International Annals of Medicine Vol1;2(5);2018 package for social sciences (SPSS; version 22, IBM, USA) software. All tests were performed at 95% confidence interval (C.I) and 5% error was used for statistical inferences. Statistical difference amongst treatment was determined using ANOVA, followed by tukey’s post hoc for analysis between group differences. Chi square was used to determine association between age, gender and cholera infection.

3. RESULTS Social Demographic Distribution of Participants A total of 109 participants were involved upon which 109 stool samples were collected. Demographic data on both gender and age were collected. Majority of the study participants 23.9% (n=26), were aged between 26-30 years followed by 0-5 years 17% (n=19). The least population of study participants

Isolate

1.8% (n=2), were aged 41-45 years whereas highest age group representing ˃ 50 years of age had only 5.5% (n=6) participants. On the other hand, gender distribution amongst study participants described highest population amongst males at 60.6% (n=66), while females accounted for 39.4% (n=43) of study population. The highest population comprised of males at 60.6% (n=66) while female were 39.4% (n=43) of the total population. Prevalence of V. cholerae serotypes isolated from stool samples Amongst 109 stool samples analyzed for V. cholerae infection (n=34; 31.2%) were positive for Inaba serotype. On the other hand, (n=75; 68.8%) of the stool samples tested negative for V. cholerae. Interestingly, none of ogawa and hikojima serotypes were isolated from the study stool samples.

Table 1: Prevalence of V. cholerae serotypes isolated from stool samples. Frequency Valid Percentage

V. cholerae Inaba serotype isolated (Positive samples) No V. cholerae isolated (Negative samples)

34

31.2

Cumulative Percentage 31.2

75

68.8

100.0

Total

109

100.0

Distribution of V.cholerae Inaba serotype infection amongst different age groups and gender Out of the 34 V. cholerae Inaba serotype cases in the study, males were more affected at 24 (70.6%), while females accounted for 10 (29.4%). On the other hand,

the most affected age group represented 26-30 years (32.4%) while least affected age group was 11-15 years (2.9%). There was no association between gender and cholera infection (table 2; p˃0.05).

Table 2: Association between gender and isolation of V. cholerae Chi-Square Tests

Pearson Chi-Square Continuity Correction Likelihood Ratio Fisher's Exact Test Linear-by-Linear Association McNemar Test N of Valid Cases

Value

df

Asymp. Sig. (2sided)

2.873 2.206 2.944

1 1 1

.090 .138 .086

2.847

1

Exact Sig. (2sided)

Exact Sig. (1 sided)

0.098

0.068

.092 0.000

109

Ho= there is no association between gender and isolation of V.cholerae

The most affected age category was 26-30 (32.4%) followed by 0-5(14.7%) and 36-40 (11.8%). The least affected age group was 11-15(2.9%). The mean age most affected 24.6 and mode age group is 26-30. The

highest age group of 46-50 had 5.9% (n=2) infection with V. cholerae. There was an association between age of the study participants and cholera infection (Table 3; p≤0.05).

To Cite This Article: Lameck Nyangena Ontweka, Margaret W. Muturi, Francisco Luquero. Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan. International Annals of Medicine. 2018;2(5). https://doi.org/10.24087/IAM.2018.2.5.448

International Annals of Medicine Vol1;2(5);2018 Table 3: Association between age category and V. cholerae infection Chi-Square Tests

Pearson Chi-Square Continuity Correction Likelihood Ratio Fisher's Exact Test Linear-by-Linear Association McNemar Test N of Valid Cases

Value

Df

Asymp. Sig. (2sided)

10.558 9.175

1 1

.001 .002

11.786

1

.001

10.461

1

.001

Exact Sig. (2sided)

Exact Sig. (1-sided)

.001

.001

.314 109

Ho= there is no association between age category and isolation of V. cholerae

Antimicrobial Susceptibility Patterns of Vibrio cholerae Inaba Serotype Isolates Results revealed that of the 34 V. cholerae Inaba serotype isolates, 15 (44.1%) were resistant, 7 (20.6%) intermediate and 12 (35.3%) sensitive to chloramphenical. Interestingly, no antimicrobial resistance was observed against ciprofloxacin and norfloxacin, with 4 (11.8%) V. cholerae Inaba serotype isolates exhibiting intermediate resistance while 30 (88.2%) were sensitive to both aforementioned drugs respectively. On the contrary, all the 34 (100%) V. cholerae Inaba serotype isolates were resistant to Nalidixic acid. Remarkably, V. Antibiotics

cholerae Inaba serotype isolates revealed utmost sensitivity to ceftriaxone, accounting for 31 (91.2%). The profile of antimicrobial responses of the isolates against ampicilin accounted for 8 (23.5%) resistant, 14 (41.2%) intermediate and 12 (35.3%) sensitive. Contrastingly, there was no V. cholerae antimicrobial resistance observed against tetracycline, however 4 (11.8%) and 30 (88.2%) showed intermediate and sensitive outcomes respectively. V.cholerae Inaba serotype isolates exhibited highest intermediate susceptibility at 25 (73.5%) to Nitrofurantoin compared to other antimicrobial agents tested (table 4).

Table 4: Antimicrobial susceptibility patterns of V. cholerae Inaba serotype isolates Resistant (R) Intermediate (I) Sensitive (S) N (%)

N (%)

N (%)

Chloramphenicol Ciprofloxacin

15(44.1%) 0

7 (20.6%) 4 (11.8%)

12 (35.3%) 30 (88.2%)

Norfloxacin

0

4 (11.8%)

30 (88.2%)

Nalidixic acid Ceftriaxone Ampicilin

34(100%) 0 8(23.5%)

0 3 (8.8%) 14 (41.2%)

0 31(91.2%) 12(35.3%)

Tetracycline

0

4 (11.8%)

30 (88.2%)

Nitrofurantoin

2(5.9%)

25 (73.5%)

7(20.6%)

Trimethoprim and Sulfamethoxazole (Cotrimoxazole)

7(20.6%)

21 (61.8%)

6(17.6%)

N: represents number of V.cholerae Inaba serotype isolates

4. DISCUSSION Out of 34 cholera positive patients, the most affected gender was male 24 (70.6%) while female were less affected 10(29.4%). This finding agrees with the study

conducted by Mutonga et al. [11], in Kenya which indicates that out of 125 cholera cases, the number of infections was higher in males 51% (n=5676) compared to females 49% (n=5449) [11]. Yet another study conducted in Uganda showed that cholera

To Cite This Article: Lameck Nyangena Ontweka, Margaret W. Muturi, Francisco Luquero. Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan. International Annals of Medicine. 2018;2(5). https://doi.org/10.24087/IAM.2018.2.5.448

International Annals of Medicine Vol1;2(5);2018 occurred 1.6 times more in males 61% (n=141) than females 39% (n=89) [12]. The most affected age group with cholera was 26-30 years (32.4%) followed by 0-5 years (14.7%) while the least affected accounted for 41-45 (1.8%). These findings are similar to a study done in slums of Kolkata, India by Sur et al. 2005 [13], indicating that the burden of cholera was greatest among those less than 2 years [13]. Likewise, a study by Jacqueline et al. [14] revealed that the infection rates were highest in children below 5 years of age [14]. Cumulatively, the current findings are parallel with those of a study conducted in Uganda which reported that the majority of cholera infections occurred in the age group below 45 years [12]. The cholera incidences in Juba study area may be attributed humanitarian crisis such as war leading to poor social economic status in the region. Multiple risk factors in war-torn regions include inadequate disease surveillance and response systems, poor infrastructure, dysfunctional health systems and disruption of disease prevention and control programs [15]. The study shows that cholera is resistant to nalidixic acid. A similar scenario was observed by Tran et al (2012) [16] who reported 100% V. cholera resistant to nalidixic acid. Additionally, 100 % resistance to nalidixic acid was also observed in a study by Miwanda et al (2015) [17]. Husna et al. reported high resistant of V. cholera (41.6%) against chloramphenicol [18] displaying similar response patterns of V. cholerae to findings of this study. This

study observed cholera sensitivity to tetracycline, ciprofloxacin and norfloxacin. Overly, these results compared with the study carried out in Tanzania which showed high V. cholera sensitivity to ciprofloxacin 98.6% and tetracycline 93.6% [19]. In a different study conducted in Papua New Guinea V. cholera showed high sensitivity to ciprofloxacin (99%) and norfloxacin (100%) [20].

5. CONCLUSION Sero-group V. cholerae O1 Inaba had high prevalence of 100% (n=34). Sero type Ogawa was not detected in this study area. There was a high prevalence of cholera amongst males than females. The study revealed that individuals between the age of 26-30 years had the highest burden of Cholera. Finally, there was no V. cholerae resistance to ciprofloxacin, norfloxacin and tetracycline.

6. RECOMMENDATION Surveillance of serotypes and antimicrobial resistance should be advanced to monitor future trends of serotype switching and drug resistance in Juba, South Sudan. ACKNOWLEDGEMENTS The authors express gratitude to Cholera Treatment Centers (CTCs) in JTH, Munuki and Gumbo in Juba, South Sudan where samples were obtained as well as the Public Health Laboratory (PHL) where study test assays were conducted. Additionally, authors thank Kenyatta University for facilitating space and accessibility to online databases used to gather relevant information for this study.

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To Cite This Article: Lameck Nyangena Ontweka, Margaret W. Muturi, Francisco Luquero. Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan. International Annals of Medicine. 2018;2(5). https://doi.org/10.24087/IAM.2018.2.5.448

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To Cite This Article: Lameck Nyangena Ontweka, Margaret W. Muturi, Francisco Luquero. Prevalence and Antimicrobial Susceptibility Profiles of Vibrio Cholerae Serotypes in Juba, South Sudan. International Annals of Medicine. 2018;2(5). https://doi.org/10.24087/IAM.2018.2.5.448