Prevalence, Risk factors and Antibiogram analysis of Nosocomial Infection in Tertiary Care Hospital of Rawalpindi, Pakistan

Abstract


Introduction
Nosocomial Infections (NIs) or Hospital-acquired infection (HAIs), or Healthcare-associated infections (HCAIs) are those infections that occur within 48 hours of hospitalization or during 30 days after taking treatment from a hospital (1,2).A worldwide survey conducted by WHO shows that 1.4 million peoples remain infected from Nosocomial infection at any time, responsible for 80,000 deaths per year (3,4).The developing countries are 2 to 20 times more susceptible to NIs, accounts for 10% infections than developed countries having 7% of us (5,6).Various negative impacts of NIs includes an excess amount of nancial loss for the patients and their family due to treatment di culties and prolonged hospital stay, increasing antimicrobial resistance, long-term disabilities, and increase death ratio (7).Diagnosis of us is still a global problem because it relies on multiple criteria, not on a single diagnostic test, and a lack of attention of national systems of continuous surveillance.Nosocomial Infections are founds in every setting, from ambulatory to long term hospital care, an alternative problem that no institution or country can claim to have solved yet (7).NIs is a global health-care problem; however, the global burden is unknown due to a lack of reliable diagnostic data (7).NIs are 2-5 times more prominent inside the intensive care unit (ICU) than in the general population (8) responsible for inducing morbidity and mortality, which is a matter of grave concern today (9).
Various factors such as immune status of the patients, the bacterial population at the infection site, mechanism of action of antibiotics, the quantity of antibacterial reach to the bacterial community (14)(15)(16)(17), induce usage of invasive materials, substandard infection control strategies, the congested environment of the hospitals and over the counter antibiotic use leads to the development of high antimicrobial resistance, especially in developing countries (18).According to a large surveillance study, around 70% of the ICU admitted patients are using antibiotics either as prophylaxis or for treatment purposes (19).
In the recent era, most of the antibiotics become non-effective against bacterial infections leads to the failure of routine treatment (20), induces patient morbidity, mortality, and healthcare-related expenses (21,22).Keeping the above literature in view, the present study was designed to analyze the various hematological, physiological changes, and antimicrobial pro les among ICU-admitted hospital-acquired infection patients.

Methodology Ethics Statement and Informed Consent
The ethical review committee of Rawalpindi Medical University approved the current study (S/No.12-13/RMU-2019).The patients were informed about the research study before sample collection.A written consent form consists of name, age, gender, date of admission, clinical settings, the reason for hospitalization, type of pathology, type of infection, and the number of days spent in the hospital before admission into ICU and start of the rst nosocomial infection were lled appropriately.

Study design
The current study was carried out between 1st February 2019, and 31st January 2020 at District headquarter Hospital a liated with Rawalpindi Medical University, Rawalpindi, Pakistan.During this study, we included all the patients admitted for more than 48 hours to the Intensive Care Unit (ICU), with no signs of bacterial colonization at the time of admission.Those patients who don't ful ll the above criteria were excluded from the study.The suspected patients of nosocomial infection were clinically examined by physicians to exclude community-acquired infection.

Sample Collection and Processing
A total of 120 patients were studied who were admitted to the ICU of the hospital, among which 30/120 (25%) patients developed at least one nosocomial episode.Various samples, including 5cc of blood, wound swab, and 10-20ml of urine in a sterile, dry, wide-necked, leak-proof container were collected using standard procedures described by Horan et al. (23).The specimens were labeled with the patient's identi cation, packed and transported within 30 minutes of the collection in a cold box to the pathology and microbiology Laboratory for further analysis.

Microbial analysis and Phenotypic Characterization of the Isolates
Samples were inoculated on Blood agar (Oxoid, UK), MacConkey agar (Oxoid, UK), and of Cystine Lactose-Electrolyte-De cient Agar (CLED) agar followed by incubation of the plate aerobically at 35-37 °C for 24 hours.The entire bacterial isolates were phenotypically characterized through their culture uniqueness and biochemical tests as earlier by Cheesbrough, (2006).Brie y, various differential and selective culture media (Oxoid, Ltd., UK), like Blood agar, Chocolate (heated blood) agar, and MacConkey agar, were used for inoculation and investigation of bacterial isolates.Various bacterial isolates were characterized through their cellular morphology, the morphology of their colony, and colonial pigmentation.Bacterial Species were characterized through various biochemical tests like catalase, oxidase, coagulase, urease, and motility tests.After overnight incubation at 37 0 C, the reading of the culture were performed by two senior medical microbiologists.

Statistical Analysis
For statistical logistic multivariate regression analysis, SPSS for Windows version 16.0 (SPSS Inc., Chicago, IL, USA) was used.P values < 0.05 were considered signi cant.
MRSA (50%) was the most frequent isolate followed by P.auroginosa (25%) found in surgical site infection, while Klebsiella oxytoca (27.7%) followed by 18.18% of Serratia liquefaciens and Serratia marcescens each was isolated from UTI. Klebsiella pneumonia (100%) was the only bacterial isolate found in respiratory tract infection.Among bloodstream infections, Klebsiella pneumonia (37.5%) was the most frequent isolate, followed by 25% of P.auroginosa and S.aureus each, as shown in the table 3.7.

Discussion
Nosocomial Infections (NCIs) or Hospital Acquired Infections (HAIs) are the leading cause of public health issues worldwide with variation in prevalence rates (26).The main strategies for managing these infections are the source and understanding of the conditions, the pathogens involved in HAI, and its risk factors (27).
The majority of the ICU patients experiences nosocomial bloodstream infections, total leukocytes count (TLC) and C-reactive protein (CRP) play a pivotal rule in the diagnosis of these infections (28-31).The current study observed that 60% of the nosocomial patients have leukocytosis that describes the possible important role of leukocytosis in nosocomial infection, as describes earlier (28-31).Lymphopenia and risk of infection are poorly studied; however, according to a report, there is a 2.4-fold increased risk of lower respiratory tract infection and urinary tract infection with lymphopenia (32).So for another study at ICU reports that lymphopenia at admittance was associated with a 1.6-fold increased risk of infection (33).Similarly, in the general population, lymphopenia was associated with an increased risk of hospitalization due to conditions like sepsis, endocarditis, disease, pneumonia, urinary tract infection, and skin infection (34).In the literature (35)(36)(37), it is well established that, in many cases, febrile neutropenic patients have bacteremia without any speci c focus (37).
Although most infections of neutropenic patients' are only clinically documented (37)(38)(39).The above-mentioned reports assist our current study, where we found 73.3% of the nosocomial patients with lymphopenia.However, discrepancies between the ndings in the present and studies (32,40, may be due to our study design that includes only nosocomial patients.The current study found Neutrocytosis in 73.3% of nosocomial infected patients as Neutrocytosis is frequently observed in the circulation and tissues during bacterial or fungal infections (42).The Neutrocytosis in the current study may be due to their important role during fungal and extracellular bacterial infections where they promote bacterial clearance through phagocytosis, production of reactive oxygen and nitrogen species (ROS/RNS), neutrophil extracellular trap (NET) formation, production of pro-in ammatory cytokin (43,44).
According to the current study, 33.3% of the nosocomial patients have thrombocytopenia, 12% have abnormal PT.In comparison, 8% have abnormal APTT; likewise, prolonged PT of 63.3% was observed in neonatal septicemia during nosocomial infection, more frequently among gram-negative infected patients (45).The deviation of our results may be because of different pathogenic microbes in the study may complicate infections by consumption coagulopathy (46), as well as the difference in the exposure to endotoxins, which may be attributed to the direct action of the endotoxins on endothelial cells or maybe an indirect result of the production of interleukin1 or tumor necrosis factor (47,48).
The liver is one of the vital organs exposed to both hepatotropic and non-hepatotropic viruses and bacteria through the portal and systemic circulation and causes liver injury, either direct invasion or indirect cytokines and toxin production (49,50).However, patients of Pneumococcal pneumonia and lobar pneumonia caused by one of the bacteria among S. pneumonia, P. aeruginosa, S. aureus, or Haemophilus in uenza sometimes show elevated concentrations of bilirubin and Serum glutamic pyruvic transaminase/serum aminotransferases (SGPT/ALT) (51).Similar reports of elevated ALT/SGPT and bilirubin have been reported in typhoid fever caused by Salmonella typhi and gastroenteritis caused by nontyphoidal Salmonella (most commonly S. enteritidis and S. Typhimurium) (52).Consistently in the current study, an elevated level of SGPT/ALT, Bilirubin, and ALP among 60%, 26.7%, and 13.3% were found respectively among nosocomial patients.The changes in liver function tests in case of nosocomial infection are may be due to hepatic injury caused by nosocomial pathogens.So for in case of liver dysfunction during systemic disease, proper microbial examination, and su cient knowledge about non-hepatotropic agents are necessary (49).
The said study nds that among nosocomial patients, 33.3% have an abnormal level of Urea.In comparison, 46.7% have an uncommon level of Creatinine; however, to our knowledge, there are no data to suggest whether or not the association of renal function test and nosocomial infection.Some previous studies report the higher risk of disease caused by MDROs, MRSA and VRE, in patients undergoing hemodialysis (53,54).The renal function test increase may be due to the kidneys complication, either direct kidney injuries or immune-mediated injuries caused by all viruses, bacteria, mycobacteria, fungus, and protozoa (55) founds in nosocomial infections.
The current study reports that upon electrolytes analysis of nosocomial patients, 26.7% have Hypernatremia, 20% have hyperchloremia, while 6.7% have Hypokalemia.The change in various electrolytes might be due to the excessive use of antibiotics, which are directly proportional to NIs.Their adverse effects may be responsible for electrolyte abnormalities such as aminoglycosides, amphotericin B, trimethoprim, and tetracycline cause electrolyte disturbance (56-58).
The prevalence of culture-con rmed nosocomial infection in the current study was 25%, which is lower than other reports of 29  73).The current study shows that imipenem was the most effective antibiotic against gram-negative isolates of Klebsiella pneumonia, K. oxytoca, Proteus spp, Serratia liquefaciens Serratia marcescens.Simultaneously, the majority of the resistance was found against Amoxicillin + Clavulanic acid, Trimethoprim/sulfamethoxazole, cefoxitin, Levo oxacin, Nor oxacin, and linezolid.Similar supporting results of sensitivity for Klebsiella pneumonia and Klebsiella oxytoca were reported (74)(75)(76).According to a study, K.pneumoniae was found resistant to all β-lactams and meropenem however susceptible to imipenem by (77) and resistance to all ß-lactams, including meropenem except imipenem was found by (78-80).A similar consistent result of sensitivity against Proteus spp was found elsewhere (81, 82).Meropenem and imipenem were the potent antimicrobials against Proteus spp.(83, 84), in contrast to the resistance against imipenem and aztreonam by (85).Resistance rates were noted highest against ceftriaxone, ceftazidime, and piperacillin/tazobactam (86).
Methicillin-Resistant Staphylococcus aureus (MRSA) was the only gram-positive isolate found in the study, highly sensitive towards Trimethoprim/sulfamethoxazole while resistant towards linezolid, Imipenem, and Cefotaxime, etc. Consistently similar reports from various countries show that around 90% of S. aureus isolated from nosocomial infections and community remain sensitive to Trimethoprim/sulfamethoxazole from the USA (98-100), Europe, Israel, and Turkey (101-103), Japan (104), Canada (105-108).A study reports higher susceptibility to amoxicillin + clavulanic acid, Doxycycline and Gentamicin, etc. (109).In contrast to the above reports, 30% of hospital-acquired MRSA in Australia, 19% in sub-Saharan Africa (110), and 85% from India (111,112) were resistant towards Trimethoprim/sulfamethoxazole.Various reports observed resistance of s.aureus towards ampicillin and penicillin, rifampicin and clindamycin, oxacillin and erythromycin (108), Azithromycin, Ceftriaxone, Ce xime and Penicillin (109), Gentamycin, Erythromycin, Levo oxacin and Tetracycline (113).The divergence in the ndings could be attributed to the mechanism of resistance like the permeability barrier, e ux pumps, mutational or recombinational changes in the target enzymes and acquired resistance by drug-resistant target enzymes in various antibiotics and alteration of the target with decreased a nity for the antibiotics (114).

Conclusion
The current study revealed that nosocomial infection is still prevalent in our hospital environment and the leading cause of drug resistance and dysfunctions of various factors like WBCs, LFTs, RFTs, electrolytes, coagulation factors and anemia, which can lead to morbidity and mortality.

Table 3 .
1. Age and Gender-Wise Distribution of Nosocomial Infections

Table 3 .
4. Liver Functional test in culture-con rmed Nosocomial Infection

Table 3 .
7. Distribution of bacterial isolates collected from patients of Nososcomial infection admitted to ICU of District Headquarter Hospital Rawalpindi, Out of all collected isolates, 100% towards imipenem was observed by Klebsiella pneumonia, Serratia liquefaciens, Proteus spp, while 75% by K. oxytoca.Besides imipenem, the sensitivity of Serratia marcescens towards ceftriaxone and amikacin were also observed.100% of Serratia marcescens isolates showed sensitivity towards ceftriaxone and amikacin.Proteus spp were found sensitive towards amikacin and gentamycin.The majority of the gram-negative isolates were found resistant towards Amoxicillin + Clavulanic acid, Trimethoprim/sulfamethoxazole, cefoxitin, Levo oxacin, Nor oxacin, and linezolid, as shown in table3.8.[Supplementary Figures 3.1, 3.2, 3.3]

Table 3 .
8. Antibiogram analysis of the entire bacterial agents isolated from Nosocomial Patients at District Headquarter Hospital Pakistan, Trimethoprim/sulfamethoxazole was a highly effective antibiotic against the gram-positive isolates (MRSA); however, all of them were resistant towards linezolid, imipenem, and Cefotaxime, etc. as shown in table3.8.[SupplementaryFigures 3.4].