Prevalence of enteric pathogens, intestinal parasites and resistance profile of bacterial isolates among HIV infected and non-infected diarrheic patients in Dessie Town, Northeast Ethiopia.

BACKGROUND: Enteric pathogens like Salmonella and Shigella species as well as intestinal parasites (IPs) are among the main causative agents of diarrhea in people with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), particularly in low income countries like Ethiopia. Antimicrobial resistance against commonly prescribed drugs has become a major global threat. This study, therefore, aimed at determining the magnitude of Salmonella, Shigella and IPs infections, their predicting factors, and antimicrobial susceptibility pattern among HIV infected and non-infected diarrheic patients in Dessie town, Northeast Ethiopia.
METHODS: A cross sectional study was conducted at three health facilities in Northeast Ethiopia between January 2018 and March 2018. Data on socio-demographic and associated risk factors were collected using structured questionnaire from 354 HIV infected and non-infected diarrheic outpatients. Fresh stool specimen was processed according to standard operating procedures. Data were entered and analyzed using SPSS version 22. Descriptive statistics was used to determine frequency, Bivariate and multivariate logistic regression analyses were performed to identify predicting factors associated with the outcome variable. P-value <0.05 were used to declare statistical significance.
RESULTS: Among 354 diarrheic patients, 112 were HIV infected and 242 were HIV non-infected. The overall prevalence of intestinal parasite and bacterial infection among HIV infected versus non-infected, respectively, was 26 (23.2%) and 8 (7.1%) versus 50 (20.7) and 16 (6.6%). Salmonella was the highest in both groups, 6 (5.4%) vs 11 (4.5%). Most prevalent parasite was C. parvum, 9 (8%) among HIV+ while E. histolytica/dispar 39 (16.1%) among HIV-. Having bloody plus mucoid diarrhea, not utilizing latrine and drinking river or spring water were factors significantly associated with bacterial infection. Whereas, being illiterate or having primary level education, diarrhea lasting for 6-10 days, CD4 level between 200-500 cells/μl, not washing hand with soap showed significant association with IPs. The bacterial isolates were 100% susceptible to Ceftriaxone and 95.4% to Ciprofloxacin, while 100% resistant to Ampicillin and Amoxicillin. MDR was observed among 19 (79.2%) isolates.
CONCLUSION: Preventing and controlling infection by enteric pathogens as well as IPs require strengthening intervention measures. The 100% resistance of isolates to commonly prescribed antibiotics calls for expanding antimicrobial susceptibility testing so as to select appropriate antimicrobial agent and prevent emergence of drug resistant bacteria.

Background

Diarrhea, as per the definition of World Health Organization (WHO) is the passage of three or more loose or liquid stools per day, or more frequently than is normal for an individual [1]. Diarrhea is still a global problem particularly in areas where access to safe water is limited and poor hygiene and sanitation are commonly practiced [2-5]. For example, in 2016, it was the eighth leading cause of death for more than 1.6 million people at global level. More than a quarter (26.93%) of the deaths occurred among under-fives. Moreover, about 90% of diarrheal deaths occurred in south Asia and sub-Saharan Africa [2].

Etiologies of diarrhea could be bacterial, viral, or parasitic. People living with human immunodeficiency virus (HIV) as well as children who are malnourished or have impaired immunity are most at risk of life-threatening diarrhea [1, 6–8]. HIV related immunosuppression, favors the occurrence of multiple opportunistic infections (OIs) which are responsible for a high morbidity and mortality. Among these OIs, intestinal parasites such as Coccidia (Cryptosporidium parvum, Isospora belli, Cyclospora sp) and amoebae (Entamoeba histolytica/dispar) are the main cause of severe chronic diarrhea [9, 10] while Salmonella and Shigella are potential enteric bacterial agents regardless of immunocompetency status of an individual [8].

With an almost 90% occurrence of HIV/AIDS in adults and children in low income countries, persistent diarrhea is associated with an 11-fold increase in mortality compared to uninfected persons [7, 8, 10].

Globally, Salmonella is one of the most frequently isolated foodborne pathogens causing diarrhea. There are an estimated 11–21 million cases of typhoid fever and approximately 128 000–161 000 deaths annually. Sub-Saharan Africa is among those harboring the majority of the cases. An estimated 2.1–6.5 million cases of invasive non typhoidal Salmonella (iNTS) disease occur annually, with the highest incidence in Africa [4, 11]. The case fatality rate is high in those with HIV infection. Salmonellosis has been estimated to be nearly 20 times as common and 5 times more bacteremia in patients with HIV/AIDS than in those without the disease [12]. On the other hand, Shigella species are among the bacterial pathogens most frequently isolated from patients with diarrhea. Globally, Shigella is estimated to cause 80–165 million cases of disease and 600,000 deaths annually [13]. The majority of the burden occurs in developing countries including Ethiopia, primarily among children and adults [14, 15].

In general, immunosuppressed individuals are more likely to be susceptible to Shigella and Salmonella infections than healthy individuals. In HIV-infected adults the rates of Gram-negative bacteria enteric infections are at least 10-fold higher than in the general population. HIV infection increases the risk of Salmonella bacteremia by about 20- to 100-fold and mortality as high as 7-fold compared to those who are not HIV infected [16].

Intestinal parasitic infections are also common among HIV patients globally and have been known to cause severe and life-threatening diarrhea [17]. Sub-Saharan Africa harbors remarkably high burden particularly among HIV positive patients [9].

Antimicrobial resistance is a global threat. Even more alarming to developing countries like Ethiopia is the development of resistance to commonly prescribed drugs leaving physicians treating patients infected with enteric pathogens like Salmonella and Shigella species with little option. Besides, access to microbiology laboratories for culture and antimicrobial susceptibility testing are limited and hence empiric treatment of microbial infections is very common. Over the past few decades Salmonella and Shigella species have become progressively resistant to most of the first-line drugs used and the prevalence of multi drug resistant strains is an important concern of treatment [18-20]. The current treatment recommendation for Salmonelosis and shigelosis by WHO [21, 22] which is also adopted by Ethiopia as well [23] is ciprofloxacin for both infections or azithromycine for salmonellosis (where there is no ciprofloxacin). Continuous monitoring of the magnitude of these infections as well as antimicrobial resistance patterns is hampered by diagnostic challenges due to lack of adequate facilities. As a result, frequent and consistent evaluation and study of the prevalence, etiologic agents, and predisposing factors of enteric fever is necessary in developing countries like Ethiopia in order to reduce its devastating effects.

This study, thus, aimed at assessing the intestinal parasitic profile as well as prevalence and antimicrobial susceptibility patterns of Salmonella and Shigella species in Dessie city, North eastern Ethiopia, where there is scarcity of such data. The study also determined the predicting factors of infections with IPs and the two enteric pathogens.

Materials and methods

Study design, area and period

A health facility based cross-sectional study was conducted from January–March 2018 at health facilities in Dessie, Northeastern Ethiopia including Dessie Referral hospital, and two randomly selected health centers, namely, Dessie health center and Banbua wuha health center. Dessie is located 401kms north of the capital city, Addis Ababa. The city is one of the fastest growing urban areas in the country and covers an area of 15.08 square kilometers. The city has one referral hospital, one general hospital (Boru Meda), six health centers (Dessie, Buanbua Wuha, Segno Gebeya, Tita, Kurkur, Meytero), four private hospitals and several private clinics. Dessie Referral Hospital is one of the largest hospitals in the region which is providing diagnosis and treatment service for the community.

Eligibility criteria

HIV infected and non-infected patients of both sexes and age greater than or equal to 15 years with diarrhea, who were willing to participate in the study were included. Those who were critically ill or patients on antibiotic or anthelminthic treatments within the previous 14 days were excluded from the study.

Data collection and laboratory processing

Structured questionnaire was used to obtain information related to socio-demographic, clinical and risk factors data from 354 HIV infected and non-infected diarrheic out patients attending the medical and ART clinics of selected health facilities conveniently. Freshly passed stool specimen was collected using pre-labeled (date, time, identification code, age), leak proof, wide mouth, sterile, screw-capped plastic container (FL Medical, Italy) and clean wooden applicator stick after appropriate instructions were given. The collected specimens were then stored in cold box and transported to Dessie Health Research Regional Laboratory and processed within two hours.

Stool examination of intestinal parasites

On a microscope slide, about 1–2 mg of stool was emulsified in a drop of normal saline (0.85% Na Cl) on the left hand side of the slide, and in Lugol’s iodine on the right side of the slide. A cover-slip was then placed on each side, and the slides were scanned under 10× and 40× objective lenses of a light microscope, as required. Saline direct smear is used mainly for detection of motility of intestinal protozoan trophozoites, which are seen in liquid or semi-liquid specimens. Iodine direct smear shows the characteristic features of the diagnostic stages in more details and formol ether sedimentation concentration technique was used for detection of cysts, ova and larvae; and additionally modified Ziehl-Neelsen technique was used for identification of Cryptosporidium and cyclospora oocysts.

Bacterial cultivation and identification

Freshly passed stool samples were placed using calibrated wire loop (0.001ml) in to Selenite F enrichment broth (Oxoid ltd, UK). After cultures were incubated overnight under aerobic condition at 37°C for 24 hours, colonies were sub cultured onto Xylose Lysine Deoxycholate (XLD) agar (Oxoid ltd, UK), Salmonella and Shigella (SS) agar (Hi media) and then incubated at 37°C for 18–24 hours. The growth of Salmonella and Shigella species was detected by their characteristic appearance on XLD agar (Shigella: red colonies, Salmonella red with a black center). Assessment was also made on SS agar (colorless colony) for Shigella and (colorless colony with black center) for Salmonella. The suspected colonies were further tested through a series of biochemical tests to identify Shigella and Salmonella species. The isolates were then characterized based on standard biochemical tests including Kligler iron agar (KIA), indole, urease, citrate utilization test, motility test, lysine decarboxylase (LDC) test [24]. Furthermore, all bacterial isolates were identified using standard clinical laboratory methods [24].

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using the standard Kirby-Bauer disk diffusion method recommended by Clinical and Laboratory Standards Institute (CLSI) [25]. Pure culture colonies of 24-hour growth were suspended in a tube with 4ml of physiological saline to get bacterial inoculums equivalent to 0.5 McFarland turbidity standards. Sterile cotton swab was dipped, rotated across the wall of the tube to avoid excess fluid and was evenly inoculated on Muller-Hinton agar (Conda ltd, USA) and then the antibiotic discs were placed on MHA plates. The following antimicrobials were used with their respective concentration: Amoxicillin (AML, 25μg), Ampicillin (AMP, 10μg), Tetracycline (TTC 30-μg), Trimethoprim-sulfamethoxazole (SXT, 25μg), Chloramphenicol (CAF, 30-μg), Naldixic acid (NAL, 30μg) Ciprofloxacin (CIP,5-μg) and Ceftriaxone (CRO, 30μg). These antimicrobial drug disks are selected based on CLSI guide and also by considering the availability and frequent prescriptions of these drugs for the treatment of Salmonella and Shigella in the study area. All antibiotic discs were from Oxoid, Ltd, UK. The plates were then incubated at 37°C for 24 hours. Diameters of the zone of inhibition around the discs were measured using a digital caliper. The interpretation of the results of the antimicrobial susceptibility tests was based on the standardized table supplied by CLSI [25] criteria as sensitive, intermediate and resistant.

Quality assurance

To generate quality and reliable data, all quality control checks were done before, during and after data collection. All the questions in structured questionnaire were prepared in a clear and precise way and translated into local language (Amharic). Data collectors were oriented how to collect data; the entire questionnaire was checked for completeness, during and after data collection by the principal investigator. Moreover, all laboratory assays were done by maintaining the quality control procedures. The raw data (the laboratory, clinical and demographic data) were checked for completeness and representativeness prior entry to the database. Standard Operating Procedures (SOPs) were strictly followed verifying that media meet expiration date and quality control parameters per CLSI guideline. Visual inspections of cracks in media or plates contamination were performed. Quality control and sterility testing were performed to check the quality of medium. Cary-Blair transport media was checked for its viability for one week by inoculating control strains and was found successful to store Salmonella or Shigella species up to one week at 2–8°C. Reference strains of Salmonella species (ATCC 13076) and Shigella species (ATCC 12022) were used as a quality control for culture and susceptibility testing throughout the study. All reference strains were obtained from Dessie Health Research Regional laboratory.

Statistical analysis

The data generated were entered in to Microsoft-Excel spreadsheet 2010 (Microsoft Cop., USA) and imported to be analyzed by Statistical Package for Social Sciences (SPSS) version 22.0. (IBM, USA). Descriptive statistics was used the compute percentages. Binary logistic regression was used to show the association of each variable with the dependent variable. Moreover, a multivariate analysis was computed to identify factors that independently influence the occurrence of the dependent variable. P-value <0.05 with 95% confidence interval was considered to declare statistical significance.

Ethical considerations

Ethical clearance was obtained from Research and Ethics Review Committee of the Department of Medical Laboratory Sciences of Addis Ababa University. Moreover, prior to commencing the study, a written informed consent was obtained from each participant. Study participants aged less than 18 years were asked an assent and written consent was taken from their parents or guardians. Confidentiality and any special data security requirements were maintained and assured. Results of the laboratory examinations were informed to physicians and the participants got their results and treatment duly as required.

Results

Socio-demographic characteristics

In this study, a total 354 study participants (112 HIV infected and 242 non-infected diarrheic patients) were investigated during the study period. The study participants were enrolled from three health facilities; of them 158 (52.2%) were from Dessie Referral Hospital, 48 (13.6%) from Buanbua Wuha Health Center, and 121 (34.2%) from Dessie Health Center with 100% response rate. Their age ranged from 15 to 86years, with mean age of 35.33±13.11 years. Distribution by sex revealed predominance of female cases in HIV infected 76(67%) and males 127 (52.5%) in HIV non infected cases. Majority of the HIV infected (73%) and non-infected (65.7%) were urban dwellers (Table 1).

Table 1

Socio-demographic characteristics of HIV infected and non-infected diarrheal patients (n = 354) from selected health facilities of Dessie Town, Northeast Ethiopia, from January to March 2018.

		HIV status		Total n (%)
		HIV positive n (%)	HIV negative n (%)
Health facilities	Dessie R Hospital	67 (59.8)	118 (77.7)	185 (52.2)
	Buanbua Wuha HC	10 (8.9)	38 (15.7)	48 (13.6)
	Dessie HC	35 (31.3)	86 (35.5)	121 (34.2)
Age (years)	15–24	19 (16.9)	66 (27.3)	85 (24)
	25–34	33 (29.5)	62 (25.6)	95 (26.8)
	35–44	38 (33.9)	55 (22.7)	93 (26.2)
	>44	22 (19.6%)	59 (24.4)	81 (22.8)
Sex	Male	36 (32.1)	127 (52.5)	163 (46)
	Female	76 (67.8)	115 (47.5)	191 (54)
Residence	Urban	82 (73.2)	159 (65.7)	241 (68)
	Rural	30 (26.8)	83 (34.3)	113 (31.9)
Education status	Illiterate	53 (47.3)	61 (25.2)	114 (32.2)
	Primary	35 (31.3)	54 (22.3)	89 (25.1)
	Secondary	17 (15.2)	65 (26.9)	82 (23.2)
	College/ University	7 (6.3)	62 (25.6)	69 (19.4)
Occupation	Civil servant	20 (17.9)	44 (18.2)	64 (18)
	Private	32 (28.6)	64 (25.4)	96 (27)
	Unemployed	49 (43.6)	84 (34.7)	133 (37.5)
	Farmer	11 (9.8)	50 (20.7)	61 (17.2)
Monthly family income (Eth birr)	< 500	37 (33)	72 (29.6)	109 (30.8)
	501–1000	28 (25)	35 (14.5)	63 (17.8)
	1001–1500	9 (8.0)	44 (18.2)	53 (15.0)
	1501–2000	17 (15.2)	32 (13.2)	49 (13.8)
	>2000	21 (18.6)	59 (24.4)	80 (22.6)

R = Referral; HC = Health center; Eth birr = Ethiopian Birr (equivalent to 1USD = 31.66 Eth birr at the time of the study)

Etiologic agents of diarrhea

The overall prevalence of enteric pathogens in stool samples of diarrheal patients was 100 (28.2%). Among these 76 (21.5%) were intestinal parasites whereas 24 (6.8%) were bacterial infections. The prevalence of intestinal parasite and bacterial infection among HIV infected was 26 (23.2%) and 8 (7.1%), respectively. Of the bacterial isolates, the predominately isolated bacteria were Salmonella 6 (5.4%) (Table 2). While the most prevalent parasite was C. parvum 9 (8.0%), followed by E. histolytica/dispar 8 (7.1%), G. lamblia 4 (3.6%) and 1 (0.9%) co-infection of C. parvum and Cyclospora cayetanensis.

Table 2

Prevalence of bacterial pathogens and intestinal parasites among HIV infected and non-infected diarrheal patients (n = 354) in selected health facilities in Dessie Town, Northeast Ethiopia, from January to March 2018.

Enteric Pathogen	HIV positive (n = 112) No. (%)	HIV negative (n = 242) No. (%)	Total (n = 354) No. (%)
Bacteria	8 (7.1)	16 (6.6)	24 (6.8)
Salmonella species	6 (5.4)	11 (4.5)	17 (4.8)
Shigella species	2 (1.8)	5 (2.1)	7 (2.0)
Intestinal parasites	26 (23.2)	50 (20.7)	76 (21.5)
E. histolytica/dispar	8 (7.1)	39 (16.1)	47 (13.3)
G. lamblia	4 (3.6)	8 (3.3)	12 (3.4)
Taenia species	2 (1.8)	0 (0)	2 (0.56)
C. parvum	9 (8.0)	0 (0)	9 (2.5)
C. parvum & C. cayetanensis	1 (0.9)	0 (0)	1 (0.3)
A. lumbricoides	0 (0)	2 (0.8)	2 (0.56)
E. vermicularis	2 (1.8)	1 (0.4)	3 (0.8)
Total	34 (30.4)	66 (27.3)	100 (28.2)
Co-infections	3 (2.8)	1 (0.4)	4 (1.1)
Shigella & C. parvum	2 (0.6)	0 (0)	2 (0.6)
Salmonella & A. lumbricoides	0 (0)	1 (0.3)	1 (0.3)
Salmonella & Taenia species	1 (0.3)	0 (0)	1 (0.3)

The prevalence of intestinal parasites and bacterial infection among HIV non-infected diarrheal patients was 50 (20.7%) and 16 (6.6%), respectively. The most prevalent intestinal parasites in this group of patients were E. histolytica/dispar 39 (16.1%) and G. lamblia 8 (3.3%) (Table 2).

Associated risk factors of Salmonella and Shigella infection

In this study, 14 independent variables were considered during the bivariate analysis of risk factors for the two enteric pathogens in diarrheal patients. Relatively higher prevalence of bacteria was found among the age group of 15–24 years, educational status of illiterates, in farmers, and among participants who do not utilize latrine, who drink river or spring water and those who had mucoid plus bloody diarrhea. In multivariate analysis, patients who had bloody plus mucoid diarrhea [AOR = 5.982, 95%CI: (1.41, 68.833), P = 0.026], who did not utilize latrine [AOR = 6.407, 95%CI: (1.139, 36.024), P = 0.035] and those who drink river or spring water sources [AOR = 8.641, 95%CI: (1.983, 37.656), P = 0.004] had a statistically significant independent association with intestinal Salmonellosis and Shigellosis regardless of HIV status (Table 3).

Table 3

Associations of risk factors with bacterial agents among study participants (n = 354) attending selected health facilities in Dessie Town, Northeast Ethiopia, January to March 2018.

Variable	Bacterial infection		COR (95% C.I)	P value	AOR (95%CI)	P Value
	Positive	Negative
Age (years)
15–24	9 (10.6)	76 (89.4)	1
25–34	6 (6.3)	89 (93.7)	0.569 (0.56–1.67)	0.31
35–44	3 (3.2)	90 (97.8)	0.281 (0.28-.074)	0.06
>44	6 (7.4)	75 (92.6)	0.676 (0.23–1.99)	0.48
Sex
Male	10 (6.0)	153 (94.)	1
Female	14 (7.3)	177 (92.7)	1.2 (0.523–2.802)	0.656
Residence
Urban	11 (4.6)	230 (95.4)	1		1
Rural	13 (11.5)	100 (88.5)	2.718 (1.178–6.275)	0.019	0.271 (0.58–1.257)	0.95
Occupation
Civil servant	1 (1.6)	63 (98.4)	1		1
Privet employee	4 (4.2)	92 (95.8)	2.74 (0.29–25.85)	0.37	0.795 (0.057–11.17)	0.865
Unemployed	12 (9.0)	121 (91.0)	6.25 (0.79–49.15)	0.08	0.682 (0.038–12.15)	0.794
Farmer	7 (11.5)	54 (88.5)	8.20 (0.97–68.49)	0.05	0.095 (0.004–2.446)	0.156
Education status
Illiterate	13 (11.4)	101 (88.6)	8.8 (1.12–68.47)	0.04	2.62 (0.127–54.06)	0.534
Primary	5 (5.6)	84 (94.4)	4.05 (0.46–35.48)	0.20	1.13 (0.058–22.16)	0.935
Secondary	5 (6.1)	77 (93.9	4.42 (.50–38.74)	0.18	3.16 (0.185–54.12)	0.427
Higher education	1 (1.4)	68 (98.6)	1		1
Monthly Family Income (ETB)
<500	14 (12.8)	95 (87.2)	3.78 (1.05–13.6)	0.04	1.974 (0.24–18.224)	0.549
501–1000	4 (6.3)	59 (93.7)	1.74 (0.375–8.07)	0.47	0.765 (0.082–7.152)	0.814
1001–1500	1 (1.9)	52 (98.1)	0.49 (0.05–4.876)	0.55	0.480 (0.032–7.261)	0.597
1501–2000	2 (4.0)	47 (96.0)	1.09 (0.176–6.78)	0.92	0.611 (0.056–6.644)	0.686
>2000	3 (3.8)	77 (96.2)	1		1
Diarrhea duration (days)
1–5	15 (5.8)	245 (94.2)	1
6–10	8 (9.6)	75 (90.4)	1.74 (0.71–4.27)	0.22
>10	1 (9.1)	10 (90.9	1.6 (0.196–13.62)	0.65
Diarrhea consistency
Watery	11 (4.4)	237 (95.6)	1		1
Mucoid	7 (13.2)	46 (86.8)	3.239 (1.208–8.902)	0.02	1.983 (0.636–6.182)	0.238
Bloody	2 (6.3)	30 (93.70	1.436 (0.304–6.793)	0.65	0.532 (0.094–3.017)	0.476
Blood + mucoid	4 (19.0)	17 (81.0)	5.07 (1.46–17.6)	0.11	5.982 (1.41–68.833)	0.026*
HIV status
Yes	8 (7.1)	104 (92.9)	1.087 (0.451–2.619)	0.853
No	16 (6.6)	226 (93.4)	1
CD4 Level
<200cells/mm3	1 (20)	4 (80)	3.778 (0.339–42.154)	0.280	1.974 (0.24–12.254)	0.749
200-500cells/mm3	4 (11.8)	30 (88.2)	3.022 (0.637–14.344)	0.164	0.765 (0.082–7.142)	0.814
>500cells/mm3	8 (7.1)	104 (92.9)	1		1
Hand wash before meal
Hand wash without soap	18 (13.8)	112 (86.2)	5.839 (1.459–17.618)	0.00	1.624 (0.345–7.638)	0.539
Hand wash with soap	6 (2.7)	218 (97.3)	1		1
Latrine utilization
Utilized	8 (2.8)	280 (97.2)	1		1
Not utilized	16 (24.2)	50 (75.8)	11.2 (4.552–27.56)	0.00	6.407 (1.139–36.024)	0.035*
Drinking water source
Pipe	7 (2.4)	279 (97.6)	1		1
Not pipe (river)	17 (25)	51 (75.0)	13.286 (5.245–33.654)	0.00	8.641 (1.983–37.656)	0.004*

Note:

*Statistically significant at P<0.05.

AOR = adjusted odds ratio, COR = crude odds ratio, 1 = reference group, 95% CI = 95% confidence interval.

Associated risk factors of parasitic infection

In this study, 12 independent variables were considered during the bivariate analysis of risk factors for intestinal parasites among diarrheal patients. Relatively higher prevalence of intestinal parasites was found among study participants who were in the age group of 15–24 years, rural residents, illiterate, unemployed, and who did not utilize latrine, who drink river or spring water, had monthly income below 500 birr and duration of diarrhea lasting 6–10 days. In multivariate analysis, participants who did not have formal education (were illiterate) [AOR = 5.8, 95%CI: (1.62, 21.04), P = 0.007], had primary level education [AOR = 3.7, 95%CI: (1.00, 13.744), P = 0.05], had 6–10 days of diarrhea [AOR = 2.039, 95%CI: (1.094, 3.80), P = 0.025], CD4 level between 200–500 cells/mm3 [AOR = 6.48, 95%CI: (2.144, 19.592), P = 0.001] and those who did not wash their hand properly with soap [AOR = 3.02, 95%CI: (1.5, 6.23), P = 0.003] had statistically significant association with intestinal parasites (Table 4).

Table 4

Bivariate and multivariate logistic regression analyses of factors associated with intestinal parasites among diarrheal patients (n = 354) attending selected health facilities in Dessie Town, Northeast Ethiopia, from Januarys to March 2018.

Variable	Intestinal parasite		COR(95% C.I)	P Value	AOR(95% C.I)	P value
	Positive	Negative
Age Group
15–24	23 (27.1)	62 (72.9)	1
25–34	15 (15.8)	80 (84.2)	0.51 (0.24–1.05)	0.067
35–44	17 (18.3)	76 (81.7)	0.60 (0.29–1.23)	0.163
>44	21 (25.9)	60 (74.1)	0.94 (0.47–1.88)	0.869
Residence
Urban	42 (17.4)	199 (82.6)	1		1
Rural	34 (30.0)	79 (70.0)	2.04 (1.21–3.44)	0.007	0.947 (0.48–1.866)	0.874
Occupation
Civil servant	5 (7.8)	59 (92.2)	1		1
Private	15 (15.6)	81 (84.4)	2.2 (0.75–6.35)	0.151	1.078 (0.332–3.495)	0.901
Unemployed	32 (24.0)	101 (76.0)	3.7 (1.4–10.12)	0.009	1.093 (0.335–3.563)	0.882
Farmer	24 (24.0)	76 (76.0)	7.6 (2.68–21.8)	0.000	2.421 (0.553–10.59)	0.240
Educational status
Higher	3 (4.3)	66 (95.7)	1		1
Illiterate	37 (32.5)	77 (67.5)	10.6 (3.2–35.9)	0.000	5.8 (1.62–21.04)	0.007*
Primary	21 (23.6)	68 (76.4)	6.79 (1.9–23.86)	0.003	3.7 (1.00–13.744)	0.050*
Secondary	15 (18.3)	67 (81.7)	4.93 (1.36–17.8)	0.015	3.5 (0.95–13.744)	0.060
Monthly Income (ETB)
<500	36 (36.3)	73 (73.7)	5.14 (2.15–12.3)	0.000	1.738 (0.60–5.031)	0.308
501–1000	14 (22.2)	49 (77.8)	2.98 (1.12–7.91)	0.028	1.276 (0.418–3.897)	0.669
1001–1500	9 (17.0)	44 (81.0	2.13 (.74–6.13)	0.160	1.072 (0.337–3.411)	0.907
1501–2000	10 (20.4)	39 (79.6)	2.67 (0.94–0.57)	0.064	1.667 (0.543–5.118)	0.372
>2000	7 (8.8)	73 (91.2)	1		1
Diarrhea duration (days)
1–5	45 (17.3)	215 (82.7)	1		1
6–10	29 (34.9)	54 (65.1)	2.56 (1.48–4.47)	0.001	2.039 (1.094–3.80)	0.025*
>10	2 (18.2)	9 (81.8)	1.06 (0.222–5.08)	0.940	1.629 (0.312–8.50)	0.563
Diarrhea consistency
Watery	45 (18.1)	203 (81.9)	1		1
Mucoid	17 (32.0)	36 (68.0)	2.13 (1.10.126)	0.025	1.249 (0.581–2.686)	0.568
Bloody	9 (28.1)	23 (71.9)	1.8 (0.765–4.07)	0.183	1.235 (0.46–3.314)	0.675
Blood + mucoid	5 (23.8)	16 (76.2)	1.4 (0.49–4.048)	0.523	1.818 (0.576–5.735)	0.308
HIV status
Yes	26 (23.2)	86 (76.8)	1.161 (0.678–1.98)	0.587
No	50(20.7)	192 (79.3)	1
CD4 Level
<200cells/mm3	3 (42.9)	4 (57.1)	4.575 (0.88–23.57)	0.69	4.058 (0.701–23.499)	0.118
200-500cells/mm3	13 (38.2)	21 (61.8)	3.776 (1.44–9.883)	0.007	6.48 (2.144–19.592)	0.001*
>500cells/mm3	10 (14.1)	61 (85.9)	1		1
Hand wash before meal
Hand wash with soap	29 (12.9)	195 (87.1)	1		1
Hand wash without soap	47 (36.2)	83 (63.8)	3.8 (2.24–6.47)	0.001	3.02 (1.5–6.23)	0.003*
Latrine utilization
Utilize	53 (18.4)	235 (81.6)	1		1
Not utilize	23 (34.8)	43 (65.2)	2.37 (1.32–4.27)	0.004	1.86 (0.501–3.836)	0.529
Water source
Pipe	56 (19.6)	230 (80.4)	1
Not pipe (river)	20 (29.4)	48 (70.6)	1.7 (0.94–3.11)	0.078

Note:

*Statistically significant at P<0.05.

AOR = adjusted odds ratio, COR = crude odds ratio, 1 = reference group, 95% CI = 95% confidence interval

Antimicrobial susceptibility pattern of bacterial isolates

All bacterial isolates from diarrheal patients (Table 5) were 100% susceptible to ceftriaxone, 95.4% to ciprofloxacin while 100% resistant to ampicillin and amoxicillin. Moreover, tetracycline, trimethoprim sulfamethoxazole/cotrimoxazole, chloramphenicol and nalidixic acid showed variable degrees of antimicrobial resistance as shown in Table 5. When data is disaggregated by species type, Salmonella isolates were 100% resistant to Ampicillin and amoxicillin while being sensitive to ciprofloxacin and ceftriaxone. On the other hand, Shigella species isolates were 100% resistant to ampicillin but susceptible to ciprofloxacin (95.6%) and ceftriaxone (100%) (Table 5).

Table 5

Antimicrobial susceptibility pattern of Salmonella and Shigella isolates among diarrheic patients attending selected health facilities of Dessie Town, Northeast Ethiopia from January to March 2018.

Antibiotics	Salmonella species N = 17(N/%)			Shigella species N = 7(N/%)			Total N = 24(N/%)
	S	I	R	S	I	R	S	I	R
AMP(10 μg)	0(0.0)	0(0.0)	17 (100)	0(0.0)	0(0.00)	7(100)	0(0.0)	0(0.0)	24(100)
AMX (20 μg)	0(0.0)	0(0.0)	17(100)	0(0.0)	0(0.0)	7(100)	0(0.0)	0(0.0)	24(100)
TTC (30 μg)	9(52.9)	6(35.4)	2(11.8)	4(57.1)	1(14.3)	2(28.6)	13(54.2)	7(29.1)	4(16.7)
CAF (30 μg)	11(64.7)	3(17.6)	3(17.6)	5(71.4)	1(14.3)	1(14.3)	16(66.7)	4(16.7)	4(16.7)
COT (25 μg)	10(58.8)	3(17.6)	4(23.5)	4(53.1)	0(0)	3(46.9)	14(58.3)	3(12.5)	7(29.2)
NAL (30 μg)	14(82.4)	2(11.8)	1(5.9)	4(53.1)	1(14.3)	2(28.6)	17(70.8)	4(16.7)	3(12.5)
CRX (30 μg)	17(100)	0(0.0)	0(0.0)	7(100)	0(0.0)	0(0.0)	24(100%)	0(0.0)	0(0.0)
CIP (5 μg)	17(100)	0(0.0)	0(0.0)	6(85.7)	0(0.0)	1(14.3)	23(95.8)	0(0.0)	1(4.2)

S = Susceptibility, I = Intermediate, R = Resistant, AMP = Ampicillin, AMX = Amoxicillin, TTC = Tetracycline, CAF = Chloramphenicol, COT = Cotrimoxazole, NAL = Nalidixic acid, CRX = Ceftriaxone, CIP = Ciprofloxacin

Multiple drug resistance patterns of the isolates

Overall, 24 (100%) bacterial isolates were resistant to at least one antimicrobial agent (Table 6). Multidrug resistance (MDR = Non-susceptible to ≥1 agent in ≥3 antimicrobial categories) [26] was seen in 19 (79.2%) of the isolates. About 76.5% of Salmonella isolates and 85.7% of Shigella isolates showed multidrug resistance for the tested antimicrobial drugs (Table 6).

Table 6

Multi drug resistance pattern of Salmonella and Shigella isolates from diarrheal patients in selected health facilities of Dessie Town, Northeast Ethiopia, from January to March, 2018.

Bacterial isolates	Total	Antimicrobial resistance pattern
		Ro	R1	R2	R3	R4	R5	R6	MDR
Salmonella	17(70.8)	0(0.0)	0(0.0)	4(23.5)	4(23.5)	7(41.1)	2(11.8)	0(0.0)	13(76.5)a
Shigella	7(29.2)	0(0.0)	0(0.0)	1(14.3)	2(28.6)	3(37.5)	0(0.0)	1(14.3)	6(85.7)b
Total	24(100)	0(0.0)	0(0.0)	5(20.8)	6(25)	10(41.7)	2(8.3)	1(4.2)	19(79.2)c

Ro = No antibiotic resistance, R1 = Resistance to one, R2 = Resistance to two, R3 = Resistance to three, R4 = Resistance to four, R5 = Resistance to five, R = Resistance to six and more drugs, MDR = Multi-drug resistant,

a Percent is computed from total number of Salmonella,

b Percent is computed from total number of Shigella isolates,

c Percent is computed from total number of isolates based on which MDR definition is applied.

Discussion

In this study the magnitude as well as predicting factors of Salmonella, Shigella and intestinal parasitic infections were determined among HIV infected and non-infected diarrheal patients at selected health facilities in Dessie Town, Northeast Ethiopia. Accordingly, intestinal parasites were detected in 23.2% of HIV infected and 21.7% HIV non-infected participants while bacterial infections were seen in 7.1% and 6.6% of HIV infected and non-infected patients, respectively.

The finding of 1.8% Shigella prevalence in HIV infected patients of the present study was relatively close to findings of 1.1% from Jimma, southwest Ethiopia [14]. However, our finding was lower compared to the 3.7% from Pune India [8] and 3.5% reported from studies done in Gondar, northwest Ethiopia [27] and 5.3% in Nigeria [28]. A study from Kampala, Uganda reported a prevalence rate of 11.1% in HIV infected children with acute diarrhea [29]. The variations noted might be due to age group, socioeconomic factor, the nature of the public water supply and seasonal variations among the various studies. Nonetheless, the observed prevalence rates in the current and other studies are expected in diarrheic patients.

The current study detected Salmonella species in 6 (5.4%) HIV infected diarrhea patients. This finding was in line with a report from Kampalla Uganda (4.3%) [29] but higher than studies done in Nigeria (1.3%) [28] and Limma Peru (2%) [30]. However; our finding was lower than studies from Jimma southwest Ethiopia (10.8%) [14]. It is likely that differences in hygienic practices and access to clean water could explain the observed variations among the studies than differences in immune status. This is supported by the findings of no significant difference between HIV infected and non-infected participants of the current study.

The prevalence of bacterial infection among HIV non-infected patients was 16 (6.6%). Among these Salmonella accounts 11(4.5%); this finding was higher than studies done in Hawassa, southern part of Ethiopia (2.5%) [18] and Gaborone, Botswana (3.0%) [31]. Moreover, this finding was higher than two studies done in Gondar which reported 1.08% [27] and 1.04% [19] while lower than the prevalence rates from Harar 11.5% [20] and Butajira 10.5% [32]. The isolation rate of Shigella species among HIV negative study participants in our study 5 (2.1%) was relatively comparable to the studies conducted in Jimma 2.3% [33] and Kampala, Uganda 3.5% [29]. Whereas the finding was lower when compared to previous studies conducted in different parts of Ethiopia including North west Ethiopia 8.7% [27], Harar 6.7% [20], Gondar 16.9% [19]; and in Gaborone, Botswana 21% [31]. In general, reports on the magnitude of these two enteric pathogens vary among studies. The discrepancy might be attributable to differences in awareness of the people about personal and environmental hygiene among different places; in the nature of the water supply system; access to health facilities and awareness regarding food borne infectious diseases.

On the other hand, intestinal parasites were detected in 23.2% of the HIV/AIDS patients and 20.6% of the HIV negative patients. Our finding was low compared to studies conducted from Addis Ababa (50.3%, 41.1%) [34], southwest Ethiopia (44.8%, 37.8%) [35], Bahir Dar (80.3%, 33.3%) [36], Hawassa (59.8%, 48.8%) [37], Cameroon (59.5%, 9.32%) [6] and Limma Peru (55%, 21%) [30] in Latin America, for HIV positive and HIV negative participants, respectively. The reports of Cameroon were low in HIV negatives and that of Lima Peru were in line with our finding. The low rate of isolation as observed in the present study might be due to the increasing awareness of the people about personal and environmental hygiene made by the health institutions and other partners.

In this study, the prevalence of cryptosporidium species was 9 (8.0%). This result was nearly similar with findings from selected ART centers of Adama, Afar and Dire-Dawa (8%) [38], southwestern Ethiopia (11%) [35], Cameroon (12.6%) [39] and Pune India (14.8%) in Asia continent [8]. This result was higher compared to reports from Dessie, Ethiopia (1.5%) [40]. However, our finding was lower compared to several studies conducted in Addis Ababa (25%) [34], Bahir Dar (43.6%) [36], Hawassa (20%) [37], in Ethiopia, Cameroon (19%) [6] and Lima Peru (20%) in Latin America [30]. This low prevalence in this study might be due to early starting of ART, geographic differences and time gap between studies; nowadays there is a better awareness of patients about ART treatment, intestinal parasite infection and their causes.

The current study tried to identify risk factors associated with Salmonella and Shigella infections. Patients having bloody mucoid diarrhea with pus were about 6 times more likely [AOR = 5.982, 95%CI: (1.41, 68.833), P = 0.026] to harbor these enteric pathogens than those with watery diarrhea. This finding agrees with similar reports from Mekelle [41] and Harar [20]. This is possibly due to the ability of those bacteria to destruct tissue, invade and replicate in cells lining the colon and rectum. The bacteria serotype is also another cause of bloody plus mucoid diarrhea in stool samples.

Latrine usage and source of drinking water were identified as predisposing factors of bacterial infection as evidenced by multivariate analysis of this study. Patients who did not utilize latrine had been about 6 times more likely to have bacterial infections of Salmonella and Shigella [AOR = 6.407 95%CI: (1.139, 36.024), (P = 0.035)] than patients who did. This could be due to open space defecation (ineffective feces disposal) which attracts flies and other insects that can transmit those diseases. This finding was supported by a study done in Mekelle which showed 7.5 times more risk among those who did not have toilet in their home [41].

Drinking water source was another factor that exposed patients to Salmonella and Shigella infections. Patients who drink river or spring water (untreated water) were about 9 times more likely to have Salmonella and Shigella infections [AOR = 8.641, 95%CI:(1.983, 37.656), (P = 0.004)] than those who drink treated pipe water. It is obvious that the bacteria mainly transmitted person-to-person by the feco-oral route, primarily by people with unhygienic hands and through drinking contaminated water. Unhygienic practices accounting for the higher likelihood of infection by these bacterial agents is also documented by the study in Mekelle city [41]. This is in line with a well-established fact that diarrhea is a global problem in areas with limited access to safe water and where poor hygiene and sanitation are commonly practiced [2]. In this study, illiterate participants were 5.8 times more likely and those at primary education were 3 times more likely to have intestinal parasites than those who attained higher education. People with no formal education and those at primary level could have limited awareness about the transmission route of intestinal parasites.

On the other hand, patients who had diarrhea elapsing for 6–10 days had more than two-fold significant association with intestinal parasite positivity [AOR = 2.039, 95%CI:(1.094, 3.80), (P = 0.025)]. Moreover, as can be expected, patients who did not wash hands properly with soap before meal had 3 times higher risk of having intestinal parasitic infection [AOR = 3.02, 95%CI:(1.5, 6.23) (P = 0.003)] than patients who wash their hands properly with soap.

Finally, drug susceptibility pattern analysis in the current study revealed resistance of all bacterial strains to at least two drugs. Of note, ampicillin, amoxicillin, cotrimoxazole and tetracycline were not effective drugs. The main reason could be the frequent use of these antibiotics in the country. Among the seventeen Salmonella species isolates, the overall rate of resistance was high for Amoxicillin and Ampicillin (100%), Tetracycline (47%), Cotrimoxazol (41%), Chloramphenicol (35.2%) and Nalidixic acid (17.6%) whereas 17 Salmonella species were 100% sensitive to Ceftriaxone and Ciprofloxacin. This finding was comparable with the results of Gondar [27] which demonstrated resistance of Ampicillin (75%), Amoxicillin (100%), Cotrimoxazole (50%), Nalidixic acid (25.0%). Such high resistance rates were reported from other parts of Ethiopia like Jimma [14] (100%) Ampicillin, (47%) Tetracycline, (26%) Nalidixic acid, and Harar [20] (both Ampicillin and Amoxicillin were 100% resistant). In contrast to our findings, the study from Sudan showed 100% sensitivity of their isolates to chloramphenicol and tetracycline, while 64% to ampicillin [42], and all isolates of Salmonella showed susceptibility to all antibiotics like Ampicillin, Tetracycline, Cotriamoxole and Chloramphenicol as reported in Gondar [19]. The time lapse between the two Ethiopian studies could partly explain the difference in the sensitivity pattern.

In this study, Shigella species showed the highest resistance to Ampicillin 7 (100%), Cotrimoxazole 3 (46%) and Amoxicillin 7 (100%). This finding has to be interpreted with care, since the numbers of isolates are small. Nonetheless, similar findings have been reported by studies done at Harar [20], Gondar [27], and Mekelle [41] which demonstrated resistance rates between 88% to 100% to both Ampicillin and Amoxicillin. This could be due to the over use of these drugs for many years. Therefore, according to this study, Ampicillin, Tetracycline, Amoxicillin and SXT are no longer effective for the treatment of Shigellosis and Salmonellosis in the study area. On the other hand, lower resistance (higher rate of sensitivity) rates were observed against Ceftriaxone and Ciprofloxacin which is an indicative of possible use of these drugs as an empiric therapy particularly in the study area. The possible justification for such low level resistance might be attributable to infrequent prescription of these drugs. Both the Ethiopian [23] and WHO recommendations [21, 22] placed Ciprofloxacin as a first-line treatment option in the management of Shigellosis. Our study provides evidence reassuring that the guideline would work well in our setting. Hence, Ciprofloxacin should be continued as a first line; and Ceftriaxone could be considered as alternative options in the treatment of Salmonella and Shigella infections.

Conclusion and recommendation

The prevalence of intestinal parasite and bacterial infection is still high in HIV infected and non-infected patients, particularly E.histolytica/dispar, Cryptosporidium species and salmonella. Unhygienic practices, unclean water source, and having bloody and mucoid diarrhea are factors independently associated with enteric bacterial infections. On the other hand, educational status, CD4 level and hand washing habit with soap before meal are factors associated with intestinal parasite prevalence. Ampicillin, amoxicillin and trimethoprim sulfamethazine are no longer effective for the treatment of Salmonella and Shigella species. Salmonella and Shigella isolates in this study are highly sensitive to Ciprofloxacin and ceftriaxone and hence could be recommended in areas where there is no culture and antimicrobial sensitivity testing facility. Of the total isolates, 76.5% and 85.7% of them were MDRs Salmonella and Shigella species, respectively. Therefore, as much as possible, early identification of diarrheal etiologic agents and antimicrobial susceptibility testing should be practiced to select the suitable antimicrobial agent and prevent the emergence as well as spread of multi drug resistant bacterial strains. Moreover, attention needs to be given to clean environment, improvement of latrine utilization, availability of safe water sources as well as health education on the transmission of enteric pathogens and IPs. In addition, emerging modified techniques in ART clinic to improve diagnosis of patients with diarrhea should be practiced.

Survey of bacterial and parasitic profile of HIV infected and non-infected diarrheal patients in health facilities of Dessie, Northeast Ethiopia.

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25 Sep 2020

PONE-D-20-27382

Prevalence of EntericPathogens,Intestinal Parasites and High Resistance rate of Bacterial Isolates to Commonly Prescribed Antibiotics in HIVInfected and Non-Infected Diarrheic Patients from selected Health Facilities in Dessie town,Northeast Ethiopia

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Reviewer #1: This is a well written manuscript that provides some useful data about pathogen prevalences and antimicrobial resistance in one area of Ethiopia. The methods seem good and well reported (the random selection of facilities is particularly commendable, as is the detailed description lab methods employed). Congratulations to the authors! I do have a few comments:

Major comments:

1) The authors need to discuss the recommended care of salmonella and shigella (both Ethiopian guidelines and WHO guidelines). For example, current WHO Shigella recommendation are to treat with Cipro. Your study suggest that guideline would work well in this region, which is reassuring. However, you then suggest Cipro as an “alternative” treatment, when in fact perhaps it should be the first line (as per guidelines)? I would suggest including the current treatment recommendation in the introduction and discussing how your results should inform these recommendation in the discussion.

2) I’m not sure that your conclusion that “the prevalence of these pathogens are high” (paraphrased) is valid. You only recruited children with diarrhea, so you were always going to see a high prevalence of enterics pathogens, and your discuss of other studies suggest that your prevalence is the same or lower than other studies have observed.

3) The discussion focus heavily on comparing your results with other work. While this is somewhat necessary for a discussion, many of the paragraphs do not appear to have an overarching point to the comparison. The discussion would be greatly improved by the authors make it clear to the reader what they should take away from each comparison. For example, in the second paragraph you compare prevalences of Shigella to other studies, what is your conclusion from this comparison?

4) In the statistical methods it would be better to identify which methods are answering which questions, i.e. you used descriptive methods to estimate the prevalences, and then model to associate exposures with the presence of pathogens. This would make it crystal clear to the reader what you are going to do.

5) You do not mention that you’re going to do a corelates of pathogen infection analysis in the introduction, you only mention looking at the prevalence. I was surprised by your correlates of infection analysis when I reached it in the results. Making this a clear goal of the paper in the introduction would be helpful.

Minor comments

1) There are some minor grammar errors, although the paper is generally well written:

Background, first paragraph, third line: “safe water is limited”

Background, fourth paragraph, seventh line: “5 times more bacteraemia”

Methods, first para, second line, “and two randomly”

Conclusion, fourth line, “On the other hand”

2) In the methods the sentence beginning “ A total of 354 HIV infected…” should be moved to the results.

3) In the antimicrobial susceptibility testing paragraph in the methods, you define the abbreviation CLSI two times.

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2 Nov 2020

Manuscript ID number:

PONE-D-20-27382

Title of paper:

Prevalence of Enteric Pathogens, Intestinal Parasites and High Resistance rate of Bacterial Isolates to Commonly Prescribed Antibiotics in HIV Infected and Non-Infected Diarrheic Patients from selected Health Facilities in Dessie town, Northeast Ethiopia

General

We thank all the reviewers for critically reviewing our manuscript which helps us to give better clarity to the paper and make it scientifically strong. We thank the PLOS ONE academic editors for their valuable comments and for giving us an opportunity to revise the manuscript.

All the questions raised by the Reviewers and Editor have been addressed; and the manuscript is modified accordingly. Moreover, the requested Editorial corrections are addressed in both the revised manuscript and the response letter. Changes are shown with track changes in the file labeled ‘Revised Manuscript with Track Changes’. The point by point response is given below.

Editor Comments:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Author’s Response:

Thanks for the significant editorial comment. The revised manuscript is now updated and meets PLOS ONE's style requirements, including those for file naming.

2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

Author’s Response:

Thank you for the comment. Copy of the questionnaire in both the original language and English is attached as Supporting Information.

3. We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [The author(s) received no specific funding for this work.]

Author’s Response:

Thank you and corrections are made as per the comment; the Funding Statement is amended and written as: “The authors received no specific funding for this work.”

Moreover, the amended statement is included within the cover letter.

4. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager.

Author’s Response:

Thank you for the valuable comment. Based up on the comment, pre-existing ORCID iD of the corresponding author is authenticated in the PLOS ONE Editorial Manager.

5. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly.

Author’s Response:

Thanks for the comment. Since the study included HIV positive patients, ethical restrictions do not allow us to share their data. However, the manuscript contains all the necessary information.

6. Please upload a copy of Supporting Information S1 Questionnaire. which you refer to in your text on page 29.

Author’s Response:

Thanks, Supporting Information S1 Questionnaire is uploaded with the revised manuscript.

7. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed.

Author’s Response:

Thanks, comment well taken; we have tried to rephrase overlapping and duplicated texts as per the comments.

Reviewer #1 comments

1. The authors need to discuss the recommended care of salmonella and shigella (both Ethiopian guidelines and WHO guidelines). For example, current WHO Shigella recommendation are to treat with Cipro. Your study suggest that guideline would work well in this region, which is reassuring. However, you then suggest Cipro as an “alternative” treatment, when in fact perhaps it should be the first line (as per guidelines)? I would suggest including the current treatment recommendation in the introduction and discussing how your results should inform these recommendations in the discussion.

Author’s Response:

Thanks for the invaluable comment. We addressed this in both sections [Ref 21-23]

2. I’m not sure that your conclusion that “the prevalence of these pathogens are high” (paraphrased) is valid. You only recruited children with diarrhea, so you were always going to see a high prevalence of enterics pathogens, and your discuss of other studies suggest that your prevalence is the same or lower than other studies have observed.

Author’s Response:

Thank you for the substantial comment. We have revised as per the comment.

3. The discussion focuses heavily on comparing your results with other work. While this is somewhat necessary for a discussion, many of the paragraphs do not appear to have an overarching point to the comparison. The discussion would be greatly improved by the authors make it clear to the reader what they should take away from each comparison. For example, in the second paragraph you compare prevalences of Shigella to other studies, what is your conclusion from this comparison?

Author’s Response:

Comment well taken and after each comparison a concluding sentence is added, we thank you very much for this.

4. In the statistical methods it would be better to identify which methods are answering which questions, i.e. you used descriptive methods to estimate the prevalences, and then model to associate exposures with the presence of pathogens. This would make it crystal clear to the reader what you are going to do.

Author’s Response:

Thank you for the valuable comment. The statistical methods part is amended accordingly.

5. You do not mention that you’re going to do a corelates of pathogen infection analysis in the introduction, you only mention looking at the prevalence. I was surprised by your correlates of infection analysis when I reached it in the results. Making this a clear goal of the paper in the introduction would be helpful.

Author’s Response:

Thanks, and we have added a clear statement stating about the goal of the paper to do corelates of pathogen infection analysis and assess possible associated risk factors at the end of the introduction part of the revised manuscript.

6. There are some minor grammar errors, although the paper is generally well written:

Background, first paragraph, third line: “safe water is limited”

Background, fourth paragraph, seventh line: “5 times more bacteraemia”

Methods, first para, second line, “and two randomly”

Conclusion, fourth line, “On the other hand”

Author’s Response:

Comments well taken and the manuscript corrected accordingly, thanks.

7. In the methods the sentence beginning “ A total of 354 HIV infected…” should be moved to the results.

Author’s Response:

Thanks; we really appreciate the comment which helped us to notice a gap. Since the result section has the similar information, we rather felt the data collection section lacks about how the data was collected. Hence, we revised the sentence and included it under this section to make the reader aware from the beginning that data was collected from the study participants conveniently.

8. In the antimicrobial susceptibility testing paragraph in the methods, you define the abbreviation CLSI two times.

Author’s Response:

Comment addressed in the method section, thanks.

Finally we thank you for your critical review of the paper.

Sincerely,

Aster Tsegaye, PhD

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

23 Nov 2020

Prevalence of Enteric Pathogens, Intestinal Parasites and Resistance Profile of Bacterial Isolates among HIV Infected and Non-Infected Diarrheic Patients in Dessie Town, Northeast Ethiopia

PONE-D-20-27382R1

Dear Dr. Tsegaye,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Iddya Karunasagar

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All reviewer comments addressed satisfactorily

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for the change you've made. I have no further comments, good luck with you continued work.

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Reviewer #1: No

4 Dec 2020

PONE-D-20-27382R1

Prevalence of Enteric Pathogens, Intestinal Parasites and Resistance Profile of Bacterial Isolates among HIV Infected and Non-Infected Diarrheic Patients in Dessie Town, Northeast Ethiopia

Dear Dr. Tsegaye:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Iddya Karunasagar

Academic Editor

PLOS ONE