Jurnal 2 Jun

Endoscopic jejunal biopsy culture: Endoscopic jejunal biopsy culture: a simple and effectivea simple and effective

method to study jejunal microfloramethod to study jejunal microflora

Chandra S, Dutta U, Noor M. T, Chandra S, Dutta U, Noor M. T, NeelamNeelam T T, Kochhar R et al, Kochhar R et al

Journal reading

Indian Society of Gastroenterology 2011

INTRODUCTIONINTRODUCTION

22

33

The small bowel / the small intestine

• 21 feet in length and begins (duodenum-jejenum-ileum).

• Normally contains bacteria colon ± 1,000,000,000 bacteria/ml of fluid small intestine (<10,000 bacteria/ ml of fluid).

• Certain species commonly found in aspirates of the jejenum E. Coli, Streptococcus, Lactobacillus, Bacteroides, and Enterococcus sp.

Pic 1. E. coli, shown in this electron micrograph, is commonly isolated in patients with bacterial overgrowth

44

Pic 2. A bacterial plate

55

ENDOSCOPY

THE JOURNALTHE JOURNAL

IntroductionIntroduction

Jejunal fluid aspirate culture Jejunal fluid aspirate culture gold standard for gold standard for studying jejunal microflora & establishing the diagnosis studying jejunal microflora & establishing the diagnosis of Small Intestinal Bacterial Overgrowth (SIBO)of Small Intestinal Bacterial Overgrowth (SIBO)..

Jejunal fluid Jejunal fluid be aspirated by be aspirated by endoscopic suction, endoscopic suction, intestinal intubation with a catheter, a capsule biopsy or intestinal intubation with a catheter, a capsule biopsy or by intraoperative bowel aspirateby intraoperative bowel aspirate..

Aspiration with a sheathed sterile catheter during Aspiration with a sheathed sterile catheter during endoscopy is most frequently used to obtain jejunal fluid.endoscopy is most frequently used to obtain jejunal fluid.

77

Microorganisms Microorganisms present in the mucus layer which present in the mucus layer which overlies, the intestinal epithelium, culture of the mucosal overlies, the intestinal epithelium, culture of the mucosal biopsy could biopsy could study the jejunal microflora study the jejunal microflora..

CCulture of the washed biopsies ulture of the washed biopsies unrepresentative. unrepresentative.

The study examineThe study examine culture of unwashed jejunal mucosal culture of unwashed jejunal mucosal biopsy to assess normal jejunal microflora and biopsy to assess normal jejunal microflora and compared it with jejunal fluid culture.compared it with jejunal fluid culture.

88

MethodsMethods

Patients Patients gastroesophageal reflux disease (GERD), gastroesophageal reflux disease (GERD), attending the gastroenterology services of a tertiary care attending the gastroenterology services of a tertiary care hospital were evaluated. Those requiring upper hospital were evaluated. Those requiring upper gastrointestinal endoscopy gastrointestinal endoscopy included included..

Patients Patients with with antibiotics, prokinetics or acid suppressants antibiotics, prokinetics or acid suppressants in 4 weeks in 4 weeks excluded. excluded.

Patients with predisposing conditions for SIBO (diabetes, Patients with predisposing conditions for SIBO (diabetes, scleroderma, hypothyroidism, and prior gastric surgery scleroderma, hypothyroidism, and prior gastric surgery excluded . excluded .

99

Enteroscopy with SIF -10, Olympus enteroscope (length 165 cm). Jejunal fluid aspirate and jejunal mucosal biopsies were obtained in the same sitting. The same procedure was repeated after 6 weeks of omeprazole (20 mg BD) therapy.

Endoscopic technique : Enteroscope sterilized in 2% glutaraldehyde (30 min) and rinsed with sterile normal saline.All patients an overnight fast, fresh sterile gloves each procedure.

1010

Jejunal fluid Jejunal fluid aspirated from proximal jejunum (50–70 cm aspirated from proximal jejunum (50–70 cm from the pylorus) using ethylene oxide sterilized 5 F metal-from the pylorus) using ethylene oxide sterilized 5 F metal-tipped ERCP cannula. 1 ml of aspirate was collected in a tipped ERCP cannula. 1 ml of aspirate was collected in a sterile syringe. The aspirate was immediately transferred sterile syringe. The aspirate was immediately transferred into 9 mL Robertson Cooked Meat (RCM) broth (Marine into 9 mL Robertson Cooked Meat (RCM) broth (Marine chemicals, Chullical, India) chemicals, Chullical, India) 1:10 dilution. 1:10 dilution.

Two mucosal biopsies were obtained from the same site Two mucosal biopsies were obtained from the same site with a sterilized multibite biopsy forceps and immediately with a sterilized multibite biopsy forceps and immediately placed in another tube containing RCM broth placed in another tube containing RCM broth transported and processed < 30 mintransported and processed < 30 min..

1111

Each biopsy was removed from the RCM broth weighed Each biopsy was removed from the RCM broth weighed on a sterile pre-weighted butter paper.on a sterile pre-weighted butter paper.

The biopsy The biopsy homogenized with 1 mL sterile saline and homogenized with 1 mL sterile saline and vortexed vortexed a uniform suspension a uniform suspension for inoculation. for inoculation. Gastric aspirate was also collected during withdrawal of Gastric aspirate was also collected during withdrawal of the enteroscope for pH estimation using micro-pHmeter the enteroscope for pH estimation using micro-pHmeter

Bacteriological analysisBacteriological analysisThe aspirated jejunal fluid The aspirated jejunal fluid transported in RCM broth transported in RCM broth (diluted 1:10, 1:100, 1:1,000) in phosphate buffer (diluted 1:10, 1:100, 1:1,000) in phosphate buffer containing 1% peptonecontaining 1% peptone..

1212

Homogenized biopsy sample diluted (1:10, 1:100, 1:1,000), Homogenized biopsy sample diluted (1:10, 1:100, 1:1,000), 20 μL of a specific dilution each of the fluid/homogenized 20 μL of a specific dilution each of the fluid/homogenized mixture mixture inoculated onto 5 separate culture plates. inoculated onto 5 separate culture plates. The total bacterial count The total bacterial count Miles and Misra method. Miles and Misra method.

The dilution which yielded countable colonies was used for computing the bacterial counts. The average number of colony counts per plate (i.e., per 20 μL) was calculated for every dilution.

1313

The culture media used for aerobic culture were chocolate agar, 5% sheep blood agar and MacConkey’s medium. The organisms were identified by standard bacteriological techniques.

Qualitative anaerobic cultures were done directly from the RCM Qualitative anaerobic cultures were done directly from the RCM tube (without dilution) onto 5% sheep blood agar containing tube (without dilution) onto 5% sheep blood agar containing haemin (5 haemin (5 μμg/ml) + menadione (1 μg/mL)g/ml) + menadione (1 μg/mL) incubated in an incubated in an anaerobic jar at 37ºC for 48 h.anaerobic jar at 37ºC for 48 h.

1414

The total colony forming units (CFU) in jejunal fluid /ml and The total colony forming units (CFU) in jejunal fluid /ml and jejunal biopsy / gram were calculated in formula.jejunal biopsy / gram were calculated in formula.

Cultures (+) for SIBO Cultures (+) for SIBO total bacterial count was total bacterial count was ≥≥1010⁵⁵ CFU / mL of fluid or per gram of biopsy tissueCFU / mL of fluid or per gram of biopsy tissue..

1515

No: of CFU per 20 μL of fluid inoculated x 1/20 x dilution x 1000

No: of CFU per 20 μL of suspension inoculated x 1/20 x dilution x 1000Weight of the biopsy in gm

Statistical methodsStatistical methodsAAnalyzed using SPSS version 10nalyzed using SPSS version 10..CCounts of total aerobes recovered from fluid and biopsy ounts of total aerobes recovered from fluid and biopsy culture.culture.The differences between baseline and postomeprazole The differences between baseline and postomeprazole therapy values of bacterial count and pH were determined therapy values of bacterial count and pH were determined Wilcoxon sign ranks test and paired t test respectively. Wilcoxon sign ranks test and paired t test respectively. An interrater reliability analysis using the Kappa statistics An interrater reliability analysis using the Kappa statistics was performed to determine consistency between fluid and was performed to determine consistency between fluid and biopsy culture for detecting SIBO, presence or absence of biopsy culture for detecting SIBO, presence or absence of any organism, aerobes and anaerobes.any organism, aerobes and anaerobes.

1616

Sample size was calculated using web based statistical Sample size was calculated using web based statistical software. With α error (5%) and β error (10%) with an software. With α error (5%) and β error (10%) with an assumed pilot correlation estimate of 0.7, minimum required assumed pilot correlation estimate of 0.7, minimum required sample size was 14.sample size was 14.

ResultsResultsEnteroscopy was performed and both jejunal fluid and Enteroscopy was performed and both jejunal fluid and mucosal biopsies were obtained in 30 patients (20 M) with mucosal biopsies were obtained in 30 patients (20 M) with GERD.Mean (SD) age of the patients was 31.3 (6.4) years. GERD.Mean (SD) age of the patients was 31.3 (6.4) years. In 18 of them, the same procedure was repeated following 6 In 18 of them, the same procedure was repeated following 6 weeks of omeprazole therapy. weeks of omeprazole therapy.

1717

Thus, 48 pairs (30 baseline and 18 postomeprazole therapy) Thus, 48 pairs (30 baseline and 18 postomeprazole therapy) of fluid and mucosal biopsies were analyzedof fluid and mucosal biopsies were analyzed..

48 pairs 48 pairs 45 pairs (culture of jejunal biopsy and jejunal 45 pairs (culture of jejunal biopsy and jejunal fluid yielded ) presence (n=27) or absence growth (n=18).fluid yielded ) presence (n=27) or absence growth (n=18).

3 pairs, growth (biopsy culture (n=2) , fluid culture (n=1) 3 pairs, growth (biopsy culture (n=2) , fluid culture (n=1) only this growth was presentonly this growth was present..

1818

1919

2020

Comparison of viable microorganismsComparison of viable microorganismsThe median (IQR) count in fluid (n=30) and biopsy (n=30) The median (IQR) count in fluid (n=30) and biopsy (n=30) culture in the patients at baseline was 0 (0–1080) CFU/mL culture in the patients at baseline was 0 (0–1080) CFU/mL and 0 (0–3200) CFU/gm, respectively. The viable aerobic and 0 (0–3200) CFU/gm, respectively. The viable aerobic bacterial counts obtained from fluid and biopsy bacterial counts obtained from fluid and biopsy cultureshowed a high degree of positive correlation cultureshowed a high degree of positive correlation Spearman’s rho=0.862; p=0.0,001)Spearman’s rho=0.862; p=0.0,001)..

The median (IQR) gastric pH increased from 3 (0.93,4.0) to The median (IQR) gastric pH increased from 3 (0.93,4.0) to 4.65 (2.77, 6.45) after six weeks of omeprazole therapy 4.65 (2.77, 6.45) after six weeks of omeprazole therapy (p=0.002). Paired cultures after omeprazole, total counts (p=0.002). Paired cultures after omeprazole, total counts increased significantlyincreased significantly..

2121

Comparison of viable microorganismsComparison of viable microorganisms105) CFU/mL to 1.2×105 (5950, 15.5×105) CFU/mL in fluid 105) CFU/mL to 1.2×105 (5950, 15.5×105) CFU/mL in fluid culture (p<0.001) and from 0 (0, 2900×105) CFU/gm to culture (p<0.001) and from 0 (0, 2900×105) CFU/gm to 2.37×105 (6500-12.5×105) CFU/gm in biopsy culture2.37×105 (6500-12.5×105) CFU/gm in biopsy culture (p<0.001).(p<0.001).

2222

SIBO was present in one of these patients at baseline. It SIBO was present in one of these patients at baseline. It was detectable on fluid culture only. After omeprazole, was detectable on fluid culture only. After omeprazole, SIBO was found in 11 (61%) patients on fluid culture SIBO was found in 11 (61%) patients on fluid culture (p=0.001) as well as on biopsy culture (p=0.001).(p=0.001) as well as on biopsy culture (p=0.001).

The microorganisms recovered from jejunal fluid and biopsy The microorganisms recovered from jejunal fluid and biopsy culture are listed in Table 1. The culture of biopsy and fluid culture are listed in Table 1. The culture of biopsy and fluid yielded similar results with regards to the distribution of both yielded similar results with regards to the distribution of both intestinal and oral flora. intestinal and oral flora.

In all the 5 patients in whom anaerobes were isolated in the In all the 5 patients in whom anaerobes were isolated in the jejunal fluid, they were also detected on jejunal biopsy jejunal fluid, they were also detected on jejunal biopsy culture.culture.

2323

Biopsy and fluid showed agreement with respect to Biopsy and fluid showed agreement with respect to presence or absence of any organismpresence or absence of any organism..

Scatter plot shows a high degree of positive correlation Scatter plot shows a high degree of positive correlation between quantitative results obtained on jejunal fluid and between quantitative results obtained on jejunal fluid and biopsy culture (n=48). Log transformation of values have biopsy culture (n=48). Log transformation of values have been done to log base 10. Spearman’s rho, 0.862, p<0.001), been done to log base 10. Spearman’s rho, 0.862, p<0.001), CFU: colony forming units. Sixteen pairs of fluid and biopsy CFU: colony forming units. Sixteen pairs of fluid and biopsy culture had no growth in eitherculture had no growth in either..

FFluid or biopsy culture and are not represented in the figure luid or biopsy culture and are not represented in the figure 0.001), aerobes (k=0.871, p<0.001) and anaerobes 0.001), aerobes (k=0.871, p<0.001) and anaerobes (k= 0.897, p<0.001).(k= 0.897, p<0.001).

2424

DiscussionDiscussionThe present study has demonstrated that the culture of The present study has demonstrated that the culture of jejunal biopsy yields results similar to fluid with respect to jejunal biopsy yields results similar to fluid with respect to presence of growth, nature of organisms, and presence or presence of growth, nature of organisms, and presence or absence of SIBO. absence of SIBO.

The bacterial counts obtained by both the methods also The bacterial counts obtained by both the methods also showed a high degree of correlation. Moreover, culture of showed a high degree of correlation. Moreover, culture of biopsy tissue is able to detect the change in nature and biopsy tissue is able to detect the change in nature and amounamount.t.

2525

In the diagnosis of SIBO, the two techniques showed high In the diagnosis of SIBO, the two techniques showed high degree of interrater reliability. degree of interrater reliability. There are several advantages of using jejunal biopsy for There are several advantages of using jejunal biopsy for culture, namely sterile biopsy forceps, which are available in culture, namely sterile biopsy forceps, which are available in all endoscopic suites; mucosal biopsies are routinely all endoscopic suites; mucosal biopsies are routinely obtainedobtained..IIn most patients undergoing endoscopy, while performing n most patients undergoing endoscopy, while performing biopsies is easier, faster, and more efficient than jejunal biopsies is easier, faster, and more efficient than jejunal fluidfluid..

2626

Moreover, the inner cup of the biopsy forceps remains Moreover, the inner cup of the biopsy forceps remains unopened during introduction and thus, theoretically has unopened during introduction and thus, theoretically has lesser chances of contamination. lesser chances of contamination. Unwashed biopsy was used for culture in the present study. Unwashed biopsy was used for culture in the present study. In the only study published in English literature, where In the only study published in English literature, where culture of unwashed biopsy was compared to luminal fluid, a culture of unwashed biopsy was compared to luminal fluid, a significant correlationsignificant correlation was obtained with respect to the total was obtained with respect to the total bacterial counts andbacterial counts and type of organisms recoveredtype of organisms recovered

2727

CCulture of biopsy was found to have a high sensitivity (90.3%), ulture of biopsy was found to have a high sensitivity (90.3%), specificity (100%), and predictive value (100%) for diagnosing specificity (100%), and predictive value (100%) for diagnosing SIBOSIBO..Previous studies investigating the bacterial flora of mucosal Previous studies investigating the bacterial flora of mucosal biopsy had used washed mucosal biopsies to remove adherent biopsy had used washed mucosal biopsies to remove adherent mucusmucus..Conflicting results regarding the similarity between luminal and Conflicting results regarding the similarity between luminal and adherent small intestinal bacterial ecologies were obtained.adherent small intestinal bacterial ecologies were obtained.These studies also reported preferential isolation of anaerobic These studies also reported preferential isolation of anaerobic organisms on culture of biopsyorganisms on culture of biopsy..

2828

Thus washed mucosal biopsy culture is possibly not Thus washed mucosal biopsy culture is possibly not representative of microbial flora compared to unwashed representative of microbial flora compared to unwashed biopsies. biopsies.

Significant increase in bacterial colony count following Significant increase in bacterial colony count following omeprazole therapy was reported in both fluid and biopsy omeprazole therapy was reported in both fluid and biopsy culture as gastric acid is the major defence mechanism culture as gastric acid is the major defence mechanism against gut bacteria proliferationagainst gut bacteria proliferation..

2929

Several other studies have also noted increase in gut Several other studies have also noted increase in gut bacterial count following omeprazole therapy. bacterial count following omeprazole therapy. Patients with predisposing conditions for SIBO were Patients with predisposing conditions for SIBO were excluded, because in addition to comparing the two excluded, because in addition to comparing the two techniques, we wanted to study the effect of omeprazole on techniques, we wanted to study the effect of omeprazole on bacterial flora and these conditions might have played a bacterial flora and these conditions might have played a confounding role.confounding role.

3030

UUssinging 1,00,000 CFU/mL as criteria for SIBO as it is the 1,00,000 CFU/mL as criteria for SIBO as it is the standard criterion for definition standard criterion for definition a after 6 weeks of fter 6 weeks of omeprazole, omeprazole, there wasthere was a significant increase in a significant increase in jejunalbacterial count and 61% were found to have bacterial jejunalbacterial count and 61% were found to have bacterial overgrowth. overgrowth. As expected, the gastric fluid pH was high after As expected, the gastric fluid pH was high after omeprazole; the reduction in gastric acid possibly explains omeprazole; the reduction in gastric acid possibly explains the higher number of gut bacteria. Achlorhydria is known to the higher number of gut bacteria. Achlorhydria is known to be a predisposing factor for SIBObe a predisposing factor for SIBO

3131

conclusionconclusion

culture of unwashed jejunal mucosal biopsy yields culture of unwashed jejunal mucosal biopsy yields organisms representative of those obtained in jejunal fluid organisms representative of those obtained in jejunal fluid culture, with respect to the counts as well as nature of culture, with respect to the counts as well as nature of organism. organism.

Culture of unwashed mucosal biopsy Culture of unwashed mucosal biopsy high sensitivity, high sensitivity, specificity, and predictive value specificity, and predictive value detecting SIBO. detecting SIBO.

Culture of mucosal biopsyCulture of mucosal biopsy useful and an easier alternative useful and an easier alternative to fluid aspirate culture to fluid aspirate culture studying jejunal microflora and for studying jejunal microflora and for diagnosis of SIBO.diagnosis of SIBO.

3232

3333

Small Intestinal Bacterial OvergrowthSmall Intestinal Bacterial Overgrowth ( (SIBOSIBO), ), = = bacterial overgrowthbacterial overgrowth = Small bowel bacterial overgrowth syndrome (SBBOS)= Small bowel bacterial overgrowth syndrome (SBBOS),, = = disorder of excessive bacterial growth in the disorder of excessive bacterial growth in the small intestine (small intestine ( coloncolon

/large bowel) rich with /large bowel) rich with bacteriabacteria, the small bowel < 10, the small bowel < 1044 /ml /ml PatientsPatients symptoms symptoms Nausea Nausea

Abdomen discomfortAbdomen discomfort Bloating Bloating

VomitingVomiting Chronic DiarrheaChronic Diarrhea Steatorrhea Steatorrhea

CostipationCostipation MalnutritionMalnutrition Weight lossWeight loss MalabsorptionMalabsorption

Gold standardGold standard aspirateaspirate from the from the jejunumjejunum101055 bacteriabacteria/ml /ml

3434