BACTERIOLOGICAL AND PARASITOLOGICAL STUDY OF ACUTE GASTROENTERITIS AMONG PATIENTS VISITING SUKRARAJ TROPICAL AND INFECTIOUS DISEASE HOSPITAL (STIDH), TEKU, KATHMANDU

BACKGROUND: Gastroenteritisis a major public-health problem in developing nations like Nepal, where communities are still ravaged by poverty, poor sanitation, poor personal hygiene, and poor water supplies.The objective of our study was to find the bacterial and parasitic agents responsible for causing gastroenteritis. METHODOLOGY: A hospital based prospective study was carried out for 3 months in Bacteriology and Parasitology section of STIDH. Stool samples received in respective section from patients with gastroenteritis were included in the study. Standard Microbiological Guidelines were employed for collection and processing of samples, followed by Isolation, Identification and Antimicrobial Susceptibility Testing of bacterial isolates. Normal saline and Iodine preparation were done for microscopic examination of parasites. RESULTS: Among 421 stool samples processed in Bacteriology section, 28(6.65%) showed bacterial growth with 15(53.57%) Shigella flexneri , 4(14.28%) Shigella sonnei and 9(32.14%) Salmonella typhimurium . Ceftriaxone and Nalidixic acid were found to be the most Sensitive and Resistant antibiotic for Shigella spp. and Salmonella typhimurium. Similarly, in parasitology section, microscopic observation of 648 stool samples showed 136(20.98%) cysts of Entamoeba histolytica , 3(0.46%) cysts of Giardia lamblia , 2(0.30%) trophozoites of Giardia lamblia , 1(0.15%) larva of Strongyloides stercoralis , 1(0.15%) ova of hookworm and 1(0.15%) ova of Ascaris lumbricoides . CONCLUSION: study

infections are found in significant amount. Hence, concerned hospital authorities and government official's needs to take strict action in contending such infections.
KEY WORDS: gastroenteritis, Shigella flexneri, Ceftriaxone, Entamoeba histolytica Background Gastroenteritis (also named as gastric flu, intestinal flu, stomach flu) is marked by inflammation of gastrointestinal tract which may be acute or chronic . Symptoms include fever, diarrhea, stomachache, nausea, vomiting, abdominal pain and cramp .
The etiological agents of gastroenteritis are bacterial, viral and parasitic. The most common bacterial agents causing gastroenteritis are Salmonella species, Shigella species, Escherichia coli and Campylobacter species. A number of parasites can also cause gastroenteritis mostly Giardia lamblia, Entamoeba histolytica, Cryptosporidium parvum .
Gastroenteritis is one of the major causes of diarrhea and vomiting among children and adults . Poor sanitation, lack of safe drinking water and lower socio-economic status contribute important cause of morbidity and mortality, and are also the major factor of disease transmission in Nepal where children are more affected than adults .
Acute gastroenteritis causes 1.5 million visits to primary care providers each year and 220,000 hospital admissions for children under the age of 5 years, a staggering 10% of all the hospital admissions of children .
In this study, we have tried to observe the bacterial and parasitic agents responsible for causing gastroenteritis. Reflecting the current status of patients suffering from gastroenteritis and the emergence of antibiotic resistance among pathogens, the findings will also help the concerned authorities to combat such infections through necessary policies and regulations in the near future.

Study area and population
After acquiring an ethical approval from Institutional Review Committee (IRC) at Nobel College, Sinamangal, the study was conducted in bacteriology and parasitology section of the hospital from August 2017 to October 2017. A total of 421 stool samples were processed for culture in the bacteriology department and 648 stool samples were processed for routine microscopic examination in the parasitology section following standard microbiological guidelines.

Bacteriological procedure
The stool samples received were cultured onto MA, XLD, TCBS and kept for overnight incubation at 37 • C.
Based upon colony morphology, gram staining, biochemical reactions (catalase, Oxidase, Methyl-Red, SIM, Urease, TSI, Citrate) organisms were identified. Serotyping was done for Salmonella and Shigella species using respective antisera (Denka Seiken Co). Non-Lactose Fermenting colonies on MA and XLD were suspected for Salmonella and Shigella species, then it was subcultured on Nutrient Agar for further investigation.

Parasitology procedure
For saline wet mount, a small quantity (about 2mg) of faeces was mixed with few drops of saline on a clean grease-free slide and then cover slip was applied to avoid formation of air bubbles. The same procedure was followed to prepare the iodine wet amount. Commercially available Normal Saline and Lugol's Iodine was used for the procedure. Slides were observed under microscope at low power magnification─10X, 20X and 40X for the detection of cysts and trophozoites of protozoa, eggs and larvae of helminths and other parasites.

Bacterial enteropathogens
Out of total 421 stool specimens examined, 28 (6.7%) were culture positive for the bacterial enteropathogens. Among the 28 isolated samples, 19 were Shigella spp. and 9 were Salmonella spp.

Figure 1: Distribution of different species of Shigella spp. and Salmonella spp.
Of the 28 culture positive samples, 11 (39.28%) were from the age group of 40-60 years, 9 from 20-40 years, 5 from 0-20 years and 2 from 60 and above age group illustrated in (Table 1).  Antibiotic susceptibility patterns of Shigella spp. and Salmonella spp.

Parasitology
Out of total 648 stool specimens─344 were male and 304 were female─144 were found to be positive for parasites, with 69 of them being male and 75 being female.

Types of parasites observed in male and female
Out  (Table 3).

Age wise distribution of parasitic infections
With 32.63% of its total patients found affected, the age group 20-30(32.63%) appeared to be most common victims of parasitic infection, whereas the age group 0-10 ranked second, with 22.22% of its total patients being infected. (Table 4).

Discussion
Gastroenteritis still accounts for a significant proportion of mortality and morbidity especially in developing countries (Khan, Singh et al. 2014). The major symptoms of gastroenteritis is diarrhea, vomiting and abdominal cramps (Ingle, Hinge et al. 2000). It is one of the most important cause of death and major public health problem in developing countries.
The study was conducted from 15 th August 2017 to 17 th October 2017 among patients visiting

Sukraraj Tropical and Infectious Disease Hospital. Keeping in view the threat of gastroenteritis in
Nepal, the present study was carried out for isolation and identification of bacterial species responsible for gastroenteritis. Out of the total 421 cases enrolled for the study, 214 were female and 207 were male, with female to male ratio being 1.03:1. Of the total positive cases, most of the bacterial species were isolated from female aged 41-50 years. Here by our study shows that adults were affected more compared to children and elder people.
In this study, Shigella species isolated were 4.51% of total cases. The pattern of serotypes found in this study was 68.42 % S. flexneri, 31.58 % S. sonnei of the total enteric isolates. This pattern of serotype was analogous to (Dhital, Sherchand et al. 2017) . The predominance of S. flexneri may be due to highly infectious nature through the oral route; an ingestion of as few or ten organisms can cause an infection. The Incidence of Shigella was commonly seen highest in summer to monsoon. The present study shows that the isolation rate was 4.51% out of 421 samples. The isolation rate was similar to the studies done by Kansakar, Dhital, Shah Sharma, and Njunda, although Khan, Huruy, Agtini, Shrestha, Wilson, Khan Singh showed that the isolation rate was comparatively more. The present study shows that Shigellosis was more common in age group 31-40. Whereas the studies conducted by Dhital, Khan, Shah, Sharma, Singh and Wilson showed that children between 1-10 age group were more susceptible to Shigellosis than adults.

Antibiotic Susceptibility Pattern of Shigella spp.
In this study, the third generation Cephalosporin were found to be more sensitive antibiotic than other antibiotic (95% of Shigella species were sensitive to Ceftriaxone and cefexime). However, 84% of isolates were resistant to Ampicillin, Nalidixic acid, Cotrimoxazole and 68% Ciprofloxacin resistant. Similar, kind of result have been shown in a study done by (Dhital, Sherchand et al. 2017). But the study carried by (Agtini, Soeharno et al. 2005) showed that the isolates were resistant to Ampicillin, Chloramphenicol and Tetracycline whereas susceptible to Nalidixic acid, Ciprofloxacin and Ceftriaxone. The study done by (Kansakar, Baral et al. 2011), (Shrestha, Malla et al. 2008) also showed that all the Shigella isolates were susceptible to Ceftriaxone and resistant to Ciprofloxacin, Ampicillin, Chloramphenicol, Cotrimoxazole which is similar to our study.
Likewise, the study of  showed that the isolates were highly resistant to Ampicillin but some strains were sensitive to Ciprofloxacin. The study conducted by (Tiruneh 2009) showed that all Shigella isolates were sensitive to Nalidixic acid and Ceftriaxone and Shigella flexneri serotype 1 showed highest resistance to Ciprofloxacin.
In this study, one of the isolates, S. flexneri was found to be ESBL-producer which accompanied ciprofloxacin resistance along with the third generation Cephalosporin. Similarly, ESBL has been reported in Shigella species from different parts of the world.

Antibiotic Susceptibility of Salmonella serovar:
Among the 421samples, 9(2.13%) of the isolates were Salmonella typhimurium. The present study shows that the isolation rate was 2.13% out of 421 samples. The isolation rate was similar to the studies done by Shah, Sharma whereas study done by Shrestha, Malla shows that the isolation rate was comparatively more.

Antibiotic susceptibility patterns of Salmonella serovar:
In this study, Salmonella serovar showed susceptibility to the third generation of Cephalosporin (Ceftriaxone), whereas Cotrimoxazole, Gentamicin, Tetracycline and Nalidixic Acid were resistant, which was similar to the study conducted by (Shrestha, Malla et al. 2008) .

Discussion of Parasitological study:
Roughly half of the world's population lives under the conditions that generate nutritional stress and parasitic disease with protozoan parasites or helminthes.  (Magar, Rai et al. 2011).The isolation rate of cyst of Giardia lamblia was 0.5%, which is less(2.6%) than the study done by (Akuffo, Armah et al. 2017).

Conclusion
The present study addresses some important issues about diarrheal infection and their most common bacterial agents at the Sukraraj Tropical and Infectious Disease Hospital. Through this study, we found that S. flexneri was an important etiological agent causing acute gastroenteritis. It is highly resistant to first-line drugs, only Ceftriaxone remains the drug of choice. However, there was one case of S. flexneri which was resistant to ceftriaxone and was an ESBL producer. With the increase in improper and imprudent use of antibiotics the chances of multi drug resistant isolates being isolated are higher, hence an alarming situation warranting proper and prudent use of antimicrobial agents and its continuous monitoring.
Intestinal parasites are worldwide in distribution. Poverty, illiteracy, unsanitary living style, usual contact with soil and consumption of vegetables, fruits and water contaminated with infected feces, walking bare foot in fecal contaminated area may play leading role in increasing rates of parasites.
Hence, there is a need for a comprehensive program to combat intestinal parasites in Nepal.