Bacteriological profile and antimicrobial susceptibility patterns of blood culture isolates among febrile patients in Mekelle Hospital, Northern Ethiopia

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Bacterial bloodstream infections are a major public health problem, which leads to high morbidity and mortality of patients. On time diagnosis and appropriate medication will be the best way to save the lives of affected ones. The aim of the present
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  Wasihun et al. SpringerPlus (2015) 4:314 DOI 10.1186/s40064-015-1056-x RESEARCH Bacteriological profile and antimicrobial susceptibility patterns of blood culture isolates among febrile patients in Mekelle Hospital, Northern Ethiopia Araya Gebreyesus Wasihun 1 , Letemichael Negash Wlekidan 1 , Senay Aregawi Gebremariam 2 ,  Tsehaye Asmelash Dejene 1 , Abadi Luel Welderufael 3 , Tadesse Dejenie Haile 4  and Saravanan Muthupandian 1* Abstract   Bacterial bloodstream infections are a major public health problem, which leads to high morbidity and mortality of patients. On time diagnosis and appropriate medication will be the best way to save the lives of affected ones. The aim of the present study was to determine the bacterial profile of bloodstream infections and their antibiotic suscep-tibility pattern in Mekelle Hospital. Cross sectional study method was carried out in 514 (269 females and 245 males) febrile patients in Mekelle hospital from March to October 2014. Standard bacteriological methods were used for blood collection, bacterial isolation and antimicrobial susceptibility pattern. Out of the total 514 febrile patients, 144 (28%) culture positive were isolated. Staphylococcus aureus  54 (37.5%), Coagulase-negative staphylococci 44 (30.6%), Escherichia coli   16 (3.1%), Citrobacter   spp. 9 (1.7%) and Salmonella typhi   8 (1.6%) were the most dominant isolates, collectively accounting for >90% of the isolates. Antimicrobial resistance pattern for gram positive and gram nega-tive bacteria was 0–83.3% and 0–100%, respectively. High resistance was seen to Trimethoprim-sulphamethoxazole 101 (70.1%), Oxacillin 65 (62.5%), Ceftriaxone 79 (58.9%) and Doxycycline 71 (49.3%). Fifty-nine percent of the isolated bacteria in this study were multi drug resistant. Most bacterial isolates were sensitive to Gentamicin, Ciprofloxacin and Amoxicillin clavulanic acid. All gram positive isolates in this current study were sensitive to vancomycin. Prevalence of bacterial isolates in blood was high. It also reveals isolated bacteria species developed multi drug resistance to most of the antibiotics tested, which highlights for periodic surveillance of etiologic agent, antibiotic susceptibility to prevent further emergence and spread of resistant bacterial pathogens. Keywords:  Prevalence, Blood culture, Bacterial isolates, Febrile patients, Antimicrobial susceptibility pattern, Multidrug resistance, Mekelle hospital © 2015 Wasihun et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the srcinal author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Background Bloodstream infection (BSI) remains one of the most important causes of morbidity and mortality globally (Zenebe et al. 2011). Tough the problem is still common in developed nations (Diekma et al. 2002), the burden is high in sub Saharan countries with 53% children mortal-ity rate (Aiker et al. 2011).Many bacteria have been reported which cause bacte-raemia with variation in distribution from place to place (Daniel et al. 2006; Asrat and Amanuel 2001; James et al. 2004; Gohel et al. 2014; Rina et al. 2007). Infection by these organisms causes prolonged patient hospitaliza-tion, increased healthcare costs and mortality rate of patients (ziamabos and Kasper 2005).reatment of bacteraemia is usually done by timely administration of appropriate antibiotics. However, since many bacterial pathogens have developed resistance to most of the antibiotics, it has become a serious health problem with many economic and social inferences Open Access *Correspondence: bioinfosaran@gmail.com 1  Department of Medical Microbiology and Immunology, Institute of Biomedical Sciences, College of Health Sciences, Mekelle University, 1871 Mekelle, EthiopiaFull list of author information is available at the end of the article  Page 2 of 7Wasihun et al. SpringerPlus (2015) 4:314 all over the world (JoAnn 2009). Researches in Ethio-pia revealed that there is high bacterial drug resistance to commonly used antibiotics mainly due to the lack of national guideline for antibiotic use, absence of good lab-oratory facilities to do antimicrobial drug susceptibility test. As a result clinicians use empirical way to treat their patients, there is also high self treatment of humans, and animals without prescription of doctors. Tese all would lead to emergence and rapid dissemination of resistance (Zenebe et al. 2011).Research findings have reported that inappropriate treatment of BSI aggravates to increased mortality of patients and emerging of drug resistance strains (JoAnn 2009; Zenebe et al. 2011; Ali and Kebede 2008). In Ethio- pia there are only a few studies on organisms involved in bloodstream bacterial infection and their susceptibility pattern (Dagnew et al. 2013; Asrat and Amanuel 2001; Zenebe et al. 2011; Ali and Kebede 2008); however, we could not get any published data in the study area and in the region as a whole. Terefore, we conducted this study to determine the common bacterial agents associated with bacteraemia and their antimicrobial susceptibility patterns in febrile patients in Mekelle hospital in igray, Northern Ethiopia. Methods Study design, study area and sampling process Prospective cross-sectional study design was used to conduct the study among febrile out patients from March to October 2014. Mekelle Hospital, which is located 783 km North of Addis Ababa, is the largest hospital in the region giving services since the last 58 years. Sample size was calculated considering 24.2% prevalence (Ali and Kebede 2008), 5% precision, and 95% confidence level; with 15% contingency, thus a total of 514 febrile patients were included. Patients who were on antibiotic within the last 2 weeks of visiting the hospital were excluded from this study. Data collection and laboratory procedures About 5 ml of venous blood for adults and 2–3 ml for children was collected aseptically using 70% alcohol and 2% tincture of iodine and transferred into a bottle con-taining 45 ml of tryptic soy broth sterile culture medium (BBL™ USA). Blood culture broths were then transported within 30 min to Ayder referral hospital microbiology laboratory and incubated at 37°C. Bacterial identification Blood culture broths were checked for sign of bacterial growth (turbidity, haemolysis, clot formation) daily up to 7 days. Bottles which showed signs of growth were fur-ther processed by gram stain and sub-culture was made onto blood agar, MacConkey agar, Manitol salt agar (all Oxoid Ltd, UK) and incubated at 37°C for 24 h. Blood culture broths with no bacterial growth after 7 days were sub-cultured before being reported as a negative result. Bacterial isolates were identified by colony morphology, gram staining reaction, biochemical tests using Cata-lase test, Coagulase test, riple Sugar Iron agar (SI) (OXOID, UK),citrate utilization test (BBL™),Urease test (BBL™) and Motility Indole Lysine (MIL) (BBL™) test using the standard procedure for bacterial identification. Antimicrobial susceptibility test Following identification of the bacterial isolates, disc dif-fusion method was done according to Clinical Labora-tory Standards Institute (CLSI) guidelines (M100 S24, 2014). Te antibiotics discs and their concentrations were: Amoxicillin-clavunilic acid; (30 µg) (OXOID, UK), Ceftriaxone, (30 µg) (BBL™),Vancomycin (30 µg) (BBL™), Oxacillin (1 µg) (BBL™ ) , Ciprofloxacin (5 µg) (BBL™), Gen-tamicin (120 µg) (BBL™), Norfloxacin (10 µg) (OXOID), Doxycycline (30 µg) (OXOID UK), Erythromycin (15 µg) (BBL™), Nitrofurantonin (300 µg) (BBL™) and rimeth-oprim-sulphamethoxazole (25 µg) (BBL™ USA). We selected these antimicrobial agents as they are available and frequently prescribed for the management of bacte-rial infections in the hospital and in Ethiopia as whole. S. aureus  and CoNS isolates which were found to be  vancomycin resistant by disk diffusion methods were sub-cultured in skim milk agar and stored at − 60°C for further Vancomycin minimum inhibitory concentration (MIC) determination by broth micro dilutions (BMD) methods (CLSI, M100 S24, 2014). Quality control Reference strains  E  . coli  (ACC 25922) and S. Aureus  (ACC 25923) were used as a control reference strains for identifications and drug susceptibility testing. Nega-tive control was done by randomly taking the prepared culture media and incubating over nigh to see for any growth. In this study multi-drug resistance was defined as simultaneous resistance to more than two antimicro-bial agents (Magiorakos et al. 2012). Data analysis SPSS version 20 software was used for statistical analysis. Chi square test ( χ 2 ) was used to determine relationship between dependent and independent variable. P value <0.05 was used to indicate significant association. Results From the total 514 febrile patients, 269 (52.3%) were females and 245 (47.7%) males. Teir age ranges from 1–80 years [mean 28.912 ±  1.46 (SD)].  Page 3 of 7Wasihun et al. SpringerPlus (2015) 4:314 wo hundred seventy-two (52.9%) of the partici-pants were in the age range of 15–29 years. A total of 144 (28%) bacterial strains were isolated. Pre-dominant isolates were S. aureus  54 (10.3%), Coag-ulase negative staphylococci (CoNS) 44 (8.5%),  E. coli  16 (3.1%), Citrobacter   spp. 9 (1.7%) and S. typhi  8 (1.6%) (able 1).More bacteria were isolated form females than males, though it was not statistically significant (P =  0.06, χ 2   =  3.47, df =  1). Te spectrum of bacteria varies with the age of patients, where 48.6% of the bacteria isolates were found in the age group of 15–29 years but not sta-tistically significant associated (P =  0.54, χ 2   =  46.4, df =  48).In vitro antibiotic susceptibility of the bacterial isolates (able 2) showed resistance for gram positive bacteria from 0 to 83%. Tirty-six (66.7%) of S. aureus  isolates were resistant to rimethoprim-sulphamethoxazole, 31 (57.4%) to Ceftriaxone, 29 (53.7%) to Doxycycline and 38 (70.4%) to Oxacillin. Tirty-six (81.8%) and 27 (61.4%) resistance was seen by CoNS to rimethoprim-sulphamethoxazole and Doxycycline, respectively. All S.aureus  and CoNS isolates were sensitive to vancomy-cin. Over all, high resistance was seen by gram positive isolates to rimethoprim-sulphamethoxazole (74%) and Doxycycline (57.7%). Amoxicillin clavulanic acid was an effective antibiotic for gram positive bacteria next to Vancomycin in our study.Antimicrobial resistance level of gram negative bacte-ria in this study was from 0 to 100%.  E. coli  was resistant to Ceftriaxone and Nitrofurantonin (60% each),  Acineto-bacter   spp. were 80% resistant to Ceftriaxone and 60% to rimethoprim-sulphamethoxazole. S. typhi  isolates were resistant to Doxycycline, rimethoprim-sulphameth-oxazole and Nitrofurantonin (75% each). Over all, high resistance was seen by gram negative isolates to Nitro-furantonin 53.3% and Ceftriaxone 46.7%. Amoxicillin cla- vulanic acid and Ciprofloxacin were, however, effective for gram negative bacteria.Antibiogram pattern of the isolates in this study showed that, S. aureus  34 (63%), CoNS 27 (61.2%), S. pyo- genes  3 (50%), Citrobacter   spp. 5 (62.5%),  E. coli  8 (53.3%) and  Acinetobacter   spp. 4 (80%) showed multiple drug resistance (MDR). In general, 59% of the isolates in our study developed multidrug resistance to different antibi-otics (able 3).MIC of Vancomycin by BMD to disc diffusion resistant CoNS and S. aureus  showed that 80% of tested isolates had Vancomycin MIC results of ≤ 1 µg/ml. wo (13.3%) S. aureus  and 1 (16.7.4%) CoNS isolate showed MIC results of ≥ 1.5 µg/ml. Only one (6.7%) S. aureus  isolates alone showed MIC results of ≥ 2 µg/ml which is intermediate resistant, where as the rest S. aureus  and all CoNS and were sensitive to vancomycin as per the 2014 guideline of CLSI. Discussion Bacteria isolation rate in this study 28%, was compara-ble with the result done in Gonder, Ethiopia, 24.2% (Ali and Kebede 2008), but higher than Addis Ababa, Ethiopia 21.4% (Asrat and Amanuel 2001), Jimma, Ethiopia (8.8%) (Zenebe et al. 2011), Gonder, Ethiopia 18.2% (Dagnew et al. 2013), Nigeria 18.2% (Nwadioha et al. 2010), Malawi 17.6% (Archibald et al. 2000), Nepal 23.1% (Amatya et al. 2007), India 9.94% (Manjula et al. 2005) and Nepal 10% (Usha and Pushpa 2007). It was, however; lower than reports from Zimbabwe 37.1% (Obi and Mazarura 1996), Gambia 34% (Philip et al. 2007) and India 40% (Neuma and Chitnis 1996). Possible explanation for the difference in prevalence of BSI across countries could be due to the difference in blood culture system, the study design, geographical location, nature of patient population, epi-demiological difference of the etiological agents, sea-sonal variations, difference in infection control policies between nations (Gohel et al. 2014; Zenebe et al. 2011; Dagnew et al. 2013).In our present study, 72.2% of infections were caused by gram-positive and 27.8% by gram-negative bacteria. Similar finding was seen from studies in Gonder, Ethiopia (69 and 31%) (Dagnew et al. 2013), Jimma, Ethiopia, (60.9 and 39.1%) (Zenebe et al. 2011) Gondar, Ethiopia (70.2 and 29.8%) (Ali and Kebede 2008), Zimbabwe (71.9 and 28.1%) (Obi and Mazarura 1996), Addis Ababa, Ethiopia (62.6 and 37.4%) (Shitaye et al. 2010).On the contrary, gram-negative bacteria have been reported as the commonest cause of bacteraemia among Table 1 Frequencies of bacterial species isolated from blood cultures of febrile patients attending Mekelle hospi-tal, Northern Ethiopia (March–October 2014) CoNS  coagulase negative staphylococcus. Bacterial speciesNo.% of the total patients (n =  514) S. aureus 5410.3CoNS448.5 E. coli  163.1 Citrobacter   spp.91.7 S. typhi  81.6 S. pyogenes 51.0  Acinetobacter   spp.40.8 S. paratyphi  20.4 P. mirabilis 10.2 Klebsiella  spp.10.2 P. aeruginosa 10.2 Total14428  Page 4 of 7Wasihun et al. SpringerPlus (2015) 4:314 febrile patients in Nigeria (69.3 and 30.7%) (Nwadioha et al. 2010), Saudi Arabia (62.2 and 33.8%) (Elbashier et al. 1998), anzania (69.7, 30.3%) (Meremo et al. 2012) and Nepal (89.19 and 10.81%) (David et al. 2004). Possi-ble explanation for the difference could be due to epide-miological difference of the etiological agents. S. aureus , CoNS,  E. coli , S. typhi , Citrobacter   spp., S.  pyogenes  and  Acinetobacter   spp. were the most common bacterial pathogens causing bacteraemia in this study. More or less similar observations have been seen in cases of bacteraemia in different countries, though, the propor-tion and prevalence of the bacterial agents varied (Dag-new et al. 2013; Asrat and Amanuel 2001; Zenebe et al. 2011; Ali and Kebede 2008; Obi and Mazarura 1996; Elbashier et al. 1998; Wisplinghoff et al. 2004). S. aureus  (37.5%) were the most commonly isolated bacteria in contrary to the other studies (Dagnew et al. 2013; Asrat and Amanuel 2001; Rina et al. 2007; Ali and Kebede 2008; Obi and Mazarura 1996) where coagu -lase negative staphylococci was the most isolated and Table 2 Antimicrobial susceptibility pattern of bacterial isolates from blood culture of febrile patients at Mekelle hospi-tal, Northern Ethiopia (March–October 2014) no. (%)  AMC   amoxicillin clavulanic acid, CRO  ceftriaxone, CN   gentamicin, DO  doxycycline, CIP   ciprofloxacin, SXT   trimethoprim-sulphamethoxazole, E   erythromycin, OX   oxacil-lin, NOR  norfloxacin, F   Nitrofurantonin, NA  not applicable. OrganismBacterial isolates resistant to each drug tested (%)AMCCROCNDOCIPSXTEOXNORF S. aureus 7 (13)31 (57.4)18 (33.3)29 (53.7)21 (38.9)36 (66.7)24 (44.4)38 (70.4)18 (33.3)NACoNS5 (11.4)22 (50)8 (18.1)27 (61.4)11 (25)36 (81.8)18 (40.9)27 (61.4)13 (29.5)NA S. pyogenes 1 (16.7)1 (16.7)04 (66.7)1 (16.7)5 (83.3)1 (16.7)01 (16.7)NA E. coli  1 (6.7)9 (60)2 (13.3)6 (40)1 (6.7)10 (6.7)NANA9 (60)4 (26.7) Citrobacter   spp.2 (25)4 (50)2 (25)4 (50)1 (12.5)3 (37.5)NANA6 (75)1 (12.5)  Acinetobacter   spp.1 (20)4 (80)02 (40)03 (60)NANA2 (40)0 S. typhi  03 (37.5)2 (25)4 (50)1 (12.5)4 (50)NANA6 (75)2 (25) S. paratyphi  01 (50)1 (50)1 (50)1 (50)1 (50)NANA11 (50) Klebsiella  spp.000000NANA1 (100)1 (100) P. mirabilis 000000NANA01 (100) P. aeruginosa 001 (100)1 (100)00NANA00 Total17 (11.8)75 (52)34 (23.6)78 (54.2)37 (25.7)89 (61.8)45 (31.3)65 (62.5)56 (38.9)10 (22.2) Table 3 Multiple drug resistance patterns of gram positive and gram negative bacteria in blood of febrile patients attending Mekelle hospital, Northern Ethiopia, (March–October 2014) CoNS  coagulase negative staphylococci  , R0  sensitive to all antibiotics tested; R1 , R2 , R3 , R4 , R5 , R6 , R7  , R8 , R9  resistant to one, two, three, four, five, six, seven, eight and nine antibiotics, respectively. OrganismsAntibiogram pattern no. (%)R0R1R2R3R4R5R6R7R8 S. aureus  (n =  54)4 (7.4)10 (18.5)5 (9.3)6 (11.1)6 (11.1)5 (9.3)8 (14.8)5 (9.3)4 (7.4)CoNS (n =  44)4 (9.1)4 (9.1)9 (20.5)9 (20.5)6 (13.6)3 (6.8)4 (9.1)4 (9.1)1 (2.3) S. pyogenes  (n =  6)–1 (16.7)2 (33.3)1 (16.7)1 (16.7)1 (16.7)––– E. coli   (n =  15  ) –5 (.3)2 (13.3)3 (20)4 (26.7)–1 (6.7)––Citrobacter spp. (n =  8)–2 (.25)1 (12.5)2 (.25)3 (37.5)–––– S. typhi   (n =  8)–2 (.25)1 (12.5)2 (.25)1 (12.5)–1 (12.5)––  Acinetobacter   spp. (n =  5)1 (20)–2 (40)–2 (20)–––– S. paratyphi   (n =  2)––1 (50)–––1 (50)–– P. mirabilis  (n =  1)–1 (100)––––––– Klebssiels  spp. (n =  1)––1 (100)–––––– P. aeruginosa  (n =  1)–––1 (100)––––– Total (144)9 (6.25)25 (17.4)24 (16.7)24 (16.7)23 (16)9 (6.25)15 (10.4)9 (6.25)5 (3.5)  Page 5 of 7Wasihun et al. SpringerPlus (2015) 4:314 in anzania, Salmonella  spp., were reported as the dominant bacteria (Meremo et al. 2012). In our study, the second most bacterial isolate was CoNS (30.6%).Tis prevalence was comparable to the study done in other parts of Ethiopia 26.1% (Ali and Kebede 2008), 33.3% (Dagnew et al. 2013), but lower than reports from Addis Ababa Ethiopia (43.3%) (Shitaye et al. 2010) and Zimbabwe (42.9%) (Obi and Mazarura 1996). Tough CoNS were mainly recognized as a contaminant until the 1970s, studies have reported an increasing incidence of infections due to these bacteria (Dagnew et al. 2013; Boisson et al. 2002).In this study,  E. coli  (10.4%) was the predominant gram negative bacteria followed by S. typhi  and Citrobacter   spp. (5.6% each). However, results from other parts of Ethiopia (Dagnew et al. 2013; Asrat and Amanuel 2001) reported that  Klebsiella  spp. and  E. coli  as the dominant isolates. In contrary to other studies (Asrat and Amanuel 2001; Zenebe et al. 2011; Ali and Kebede 2008; Usha and Pushpa 2007), we have not isolated  H. influenzae  in our study which in lines with the finding from Gonder (Dag-new et al. 2013). In our current study we found all cases of BSI with single microorganism which in lines with earlier reports (Dagnew et al. 2013; Ghanshyam et al. 2008; Angyo et al. 2001). Unlike to our study; however, septicaemia of poly-microbial aetiology were reported by other studies (Obi and Mazarura 1996; Ghanshyam et al. 2002).In this study bloodstream infection was not age dependent which was in contrast to other previous reports that showed septicaemia was relatively higher in neonates (Dagnew et al. 2013; Komolafe and Adegoke 2008; Shitaye et al. 2010).Tis may be due to small num- bers of children in our study participants.Overall the resistance of gram positive bacteria was from 0 to 83%, and for gram negative from 0 to 100% which is similar to the result from Jimma which was 0–85.7% and 0–100% for gram negative and positive, respectively (Zenebe et al. 2011). Tis was however, dif-ferent from the study result done in Addis Ababa, where the rate for gram positive bacteria ranged from 12 to 76%, and for gram negatives ranged from 8 to 46% (Asrat and Amanuel 2001) and Gonder 23.5–58.8% and 20–100% for gram positive and negative, respectively (Dagnew et al. 2013). Te increased resistant blood isolates in this study may be a signal of indiscriminate and continuous use of sub-therapeutic doses of commonly available antimi-crobials both in the veterinary and public health sectors (Zenebe et al. 2011). Tis could challenge the manage-ment of patients very difficult.Amoxicillin clavulanic acid was found to be effective against both gram positive and gram negative isolates in this study. Other studies reported Ciprofloxacin as an effective (Dagnew et al. 2013; Asrat and Amanuel 2001; Zenebe et al. 2011). Ciprofloxacin was found to be effec-tive against gram negative isolates comparable with find-ings from Ethiopia and other nations (Dagnew et al. 2013; Komolafe and Adegoke 2008; Shitaye et al. 2010). Antibiograms resistance pattern of the isolates revealed 59% them showed multidrug resistance. Tis suggests a high resistance gene pool perhaps due to gross mis-use and inappropriate usage of the antibacterial agents (Komolafe and Adegoke 2008).Except one isolate of S. aureus  which was intermedi-ate resistant, all MICs tested CoNS and S. aureus  were sensitive to Vancomycin as per the CLSI, 2014 guideline, which was similar to other studies done elsewhere (Sak-oulas et al. 2004; Soriano et al. 2008). Possible explanation for the absence of vancomycin resistant bacteria in our study is due to the fact that use of vancomycin by doc-tors is restricted in the treatment of patients in the study area and in the nation as whole. Unlike to this; however, researchers have reported vancomycin resistant strains from France (Ploy et al. 1998), Korea (Kim et al. 1998), South Africa (Ferraz et al. 2000) and Nigeria (Moses et al. 2013) showing that it is becoming a global threat.Tere are also other reports that indicated that there is an increasing vancomycin MICs over time at individ-ual institutions. However, as most clinical laboratories use automated systems to carry out susceptibility test-ing, these systems do not provide an accurate vancomy-cin MICs. Hence it has been recommended that clinical laboratories to provide accurate and reliable vancomy-cin MICs report to clinicians to help select appropriate therapy for the management of patients (Hsu et al. 2008; Steinkraus et al. 2007).In conclusion, prevalence of BSI in this current study was high. Tis study added to the knowledge of the epi-demiology of the isolates with high rates of resistance to most used antibiotics. Terefore timely investigation of bacterial flora of the bloodstream infections and moni-toring of their antibiotic susceptibility pattern is impor-tant to reduce the incidence of bloodstream infections and multi drug resistant strains. Authors’ contributions AGW was the primary researcher, conceived the study, designed, participated in data collection, laboratory work, conducted data analysis, drafted and finalized the manuscript for publication. LN, SA, TA, SM performed in data collection, analysis data, assisted in reviewed the initial and final drafts of the manuscript. TD and AL interpreted the results, and reviewed the initial and final drafts of the manuscript. All authors read and approved the final manuscript. Author details 1  Department of Medical Microbiology and Immunology, Institute of Biomedi-cal Sciences, College of Health Sciences, Mekelle University, 1871 Mekelle, Ethi-opia. 2  Department of Internal Medicine, School of Medicine, College of Health Sciences, Mekelle University, Mekelle, Ethiopia. 3  Department of Pediatric
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