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Bacterial Infections and Their Emerging Resistant Patterns in HCV


									Bacterial Infections and Their Emerging Resistant
        Patterns in HCV Patients Following
           Liver Transplantation in Egypt
                     Protocol of Thesis
   Submitted for Partial Fulfillment of the Degree of M.D.
                   in Tropical Medicine
               Yasmine Mahmoud Massoud
      M.B.B.Ch., M.Sc. (Tropical Medicine, Ain Shams University)

                       Under Supervision of

Professor Doctor/ Mohammed Fawzy Montasser
                     Ex-Dean Faculty of Medicine
                    Professor of Tropical Medicine
              Faculty of Medicine - Ain Shams University

Professor Doctor/ Mahmoud Shawky Al-Metieny
                     Professor Of General Surgery
    Director Of Ain Shams Center For Organs Transplantation (Ascot)
              Faculty of Medicine - Ain Shams University

     Doctor/ Nadia Abdel Aaty Abdel Kader
                     Lecturer of Tropical Medicine
              Faculty of Medicine - Ain Shams University

     Doctor/ Sara Mahmoud Abd El-Hakam
                     Lecturer of Tropical Medicine
              Faculty of Medicine - Ain Shams University

     Doctor/ Hany Mansour Khalil Dabbous
                     Lecturer of Tropical Medicine
              Faculty of Medicine - Ain Shams University

                          Faculty of Medicine
                         Ain Shams University

       Liver transplantation is considered the treatment of choice for patients
with end stage liver disease who have failed standard therapy. The development
of liver transplantation has revolutionized the field of hepatology and greatly
improved the outlook of patients suffering from various liver diseases. This
procedure is now applied worldwide as treatment for a large number of
irreversible acute and chronic liver diseases for which there were previously no
other treatment options (Zhu et al., 2009).

      The primary goals of liver transplantation are to prolong life and to
improve the quality of life. Thus, it is essential to optimize patient selection and
ideally time the transplant procedure so as to gain the maximum benefit
(Kanwal et al., 2005).

      Current problems encountered following OLT are surgical and Non-
surgical complications which are mostly pulmonary. Infection is the main cause
of death following OLT. Early infections, mostly intra-abdominal and
pulmonary, are bacterial or fungal (Moncorgé et al., 1989).Biliary strictures and
incisional hernias are the most common surgical complications encountered late
after liver transplantation (Porrett et al., 2009).

        More than two-thirds of liver transplant recipients have infection in the
first year after transplantation, and infection is the leading cause of death in
these patients. In addition, release of cytokines during the infection may have
other indirect and negative effects, including allograft injury, opportunistic
super infection, and malignancy (Rubin, 2002).

      There are many factors associated with an increased risk of infection,
such as disruption of the normal bacterial flora resulting from the extensive use
of broad-spectrum antimicrobial agents, prolonged or intensive antibacterial
chemo therapy, severe and chronic underlying diseases (uremia, liver cirrhosis),
nutritional status, surgical and/or invasive procedures, the hospital environment,
immunosuppressive therapy, and other factors causing immunological
deficiency (e.g. viral and fungal co-infections) (Kawecki et al., 2009)

      The most frequent infections are surgical site (16%) and urinary tract
(11%). Etiologies included bacterial (48%) and fungal (22%) causes. The most
common pathogens are Escherichia coli (20%), among which, 40% were
extended-spectrum beta-lactamase ESBL-producers, and Enterococcus spp.
(11%). More than half of the infectious episodes (58%) occurred in the first 4
months after transplantation (Garcia et al., 2009). Patients may become infected
with nosocomial, antimicrobial-resistant bacteria, including methicillin-resistant
Staphylococcus     aureus,    vancomycin-resistant      Enterococcus      faecalis,
Clostridium difficile, and antimicrobial-resistant gram-negative bacteria (Del
Pozo, 2008).

      Methicillin-resistant Staphylococcus aureus (MRSA) infection frequently
complicates the postoperative course in diseased-donor liver transplantation.
The incidence and risk factors of MRSA infection after Living-donor Liver
transplantation (LDLT), however, are unclear (Hashimoto et al., 2008).The
most common sites of MRSA infection are blood (42%), lung (38%), and
abdomen (29%) (Schneider et al., 2005).

Aim of the Work

      To study the incidence, types, risk factors and causative organisms of
bacterial infections in HCV patients following Liver Transplantation and
identify the emerging resistant strains and their proper antimicrobial therapy.

Patients and Methods

      Study design: Prospective Descriptive Follow up study.

      Participants: At least 30 Patients with eligible criteria for liver
     transplantation in Ain Shams Specialized, Wadi El Nile and Egypt Air
Hospitals will be recruited and followed up during a period of 3months
following transplantation.

 Patients will be divided into 2 groups:

       Group 1 includes patients who had a postoperative course free
   from infections.

       Group 2 includes patients who had postoperative bacterial
   infections whom will be further analyzed into a subgroup without
   emerging strains or antibiotic resistance and another subgroup who
   experienced bacterial infection with emerging or resistant strains.

 Inclusion criteria:

 - Patients with end stage liver disease who are hepatitis C virus positive
 and who are candidates for liver transplantation.

 - Age between 18-60 Years, including males and females.

 - Accepting participation in the current study and signing a written
 consent form.

 Exclusion criteria:

  -    Patients with other etiologies for end stage liver disease (HBV,

  -    Intra operative or early postoperative death.

  •    Special Follow-up items for the Current Study:

  1. All patients will be checked for the possible risk factors
  suggestive of acquiring infection.


       Demographic data as (donor age & sex, recipient age & sex).
      Child & MELD scores

      Leucopenia and thrombocytopenia

      Co-morbidities including (Diabetes, Hypertension,...)

      Nutritional status (adequately, moderately, poorly nourished)
  (Morgan et al ., 2006).

      Serum sodium

      Documented       or   suspected   SBP     and   3rd   generation
  cephalosporin administration.

      GRWR (Graft Receipient Weight Ratio).

      Degree of steatosis of the graft (<15%)

      Renal impairment.


      Total operative period

      Cold Ischemia Time.

      Warm Ischemia Time.

      Amount of transfusion of blood or blood products.

      Type of biliary anastomosis.


      ICU stay (average 3-5 days)

      Duration of central venous line and catheter insertion.
      Duration of abdominal drain

      Duration of hospital stay

      Immunosuppression given as regards: type, number & dosage
of drugs.

 2. Patients will be identified and classified into the 2 groups based on;

      a) clinical assessment

      b) laboratory results with stress on ; CBC with differential count,
         CRP, serum ferritin, liver profile abnormalities

      c) pancultures; urine, sputum, blood, ascitic, bile, stool and
         abdominal drain cultures.

  3. Infections will be defined using criteria proposed by the Center for
 Disease Control and as reported in liver transplant recipients (Garner et
 al., 1988).

*In patients with skin infections: A sample of the discharge will be taken
from the infected tissue by using a sterile dry cotton wool swab. And will
be examined by:

- Microscopic examination: by Gram smear for pus cells and bacteria.

- Culture will be done on blood agar and MacConkey’s agar (Colle et al.,

Diagnosis of wound infection is based upon the presence of
redness/induration and the presence of pus on exploration and/or positive
wound culture.

* In patients with clinical picture suggestive of urinary tract infection:
Mid-stream urine samples will be collected in a sterile dry container
(about 20 cc of urine).
- In patients with indwelling urinary catheters, urine samples will be
collected by aspiration of urine from the distal end of the catheter by a
sterile syringe after clamping the drainage tube above the catheter-tube

Samples will be examined by:

   - Microscopic examination: to detect the presence of the increased
   numbers of polymorphs (pyuria).

   - Estimation of viable bacterial colony count in uncentrifuged urine
   sample: In mid-stream urine, more than 105 CFU/ml indicates urinary
   tract infection. Less than 105 without pus cells or patient's complain
   indicate urethral contamination.

   - Culture: will be inoculated on blood agar and Mac Conkey’s agar.

   For diagnosis of urinary tract infection 1 of the following criteria
   should be present:

            Patient has at least 1 of the following signs or symptoms with
   no other recognized cause: fever (38C), urgency, frequency, dysuria,
   or suprapubic tenderness and patient has a positive urine culture, that
   is, ≥10 microorganisms per cc of urine with no more than 2 species of

   * In patients with clinical picture suggestive of pneumonia Sputum
   samples will be taken from suspected patients. Sputum samples will
   be collected in the morning before any mouth wash by coughing
   deeply in a sterile dry container. In comatosed patients, samples will
   be taken by naso-tracheal suction. The samples will be checked for:

            - Physical examination: colour, aspect and odour.
        - Microscopic examination by: Wet preparation: to detect
 epithelial cells and pus cells, Gram smear: for pus cells and to identify
 gram-positive or gram-negative organisms.

        - Culture: will be done on blood agar, MacConkey’s agar and
 chocolate agar and incubated at 37°C, both aerobically and
 anaerobically, then examined after 24 hours (Colle et al., 1996).

  Diagnosis of pneumonia is based on pulmonary infiltrates together
 with clinical symptoms suggestive of lower respiratory tract infection,
 the identification of a relevant etiologic microbial agent, and the
 absence of an alternative diagnosis during follow-up. Additional
 confirmative criteria are the response to adequate antimicrobial
 treatment or results of necropsies demonstrating the presence of

 * Blood cultures: (will be done for all patients with manifestations of
 infections). They will be collected before the start of any antimicrobial

        - Blood samples will be taken under complete aseptic condition.

        - Withdrawing 10-12 ml of blood by a sterile syringe.

        - Inserting the needle through the rubber line of the bottle cap
 and dispensing blood into each culture bottle.

        - Wiping the top of each culture bottle by an ethanol-ether swab
 and replacing the protective caps, then gently mixing the blood with
 the broth.

        - Then incubating the inoculated media (Reimer et al., 1997).

Diagnosis of Blood stream infection (BSI) is defined as the isolation
of gram-positive cocci, gram-negative rods, or any kind of fungi in the
blood culture.
      -      Antibiotic susceptibility test will be done.

      -      Cultures will be repeated after 10 days from the start of
antibiotic treatment.

      -      Infection resolution will be identified when clinical
manifestations of infection improve and cultures become negative.

 *Ascitic fluid culture (will be done for all patients with manifestations
 of bacterial peritonitis or suspected to be so) Samples will be collected
 before the start of any antimicrobial treatment.

          - Samples will be taken under complete aseptic conditions.

          - Withdrawing 2 ascitic samples, each 20 ml by a sterile

          - Chemical and bacteriological testing will be done.

 *Bile samples for those suspected to have biliary tract infection. In
 cases of suspected sepsis induced cholestasis (SIC), Cultures from
 blood, biliary tube, abdominal drains , urine, and sputum will be done
 and culture based treatment will be started accordingly.

 • Duration of the study: Will be 1 year.

 Data Management and Statistical Analysis:

     To assess risk factor status, two groups of patients will be

 Group I: Patients who did not develop infection at 3 months.

 Group II: Patients who aquired infection during the first 3 months
 post-transplant. All the pre-operative variables and post-operative
 events will be compared between the two groups using univariate and
 multivariate analysis. Student’s t-test or the Mann–Whitney U-test
will be used for quantitative data and Pearson’s chi-square or Fisher’s
exact test for categorical data. Differences will be considered
statistically significant when the p value is <0.05. Data will be shown
as mean ± SD or as median and range or as percentages. All variables
significant in univariate analysis will be analysed by a multiple
regression logistic model. The forward stepwise logistic strategy will
be applied, and variables will be included in the model if the log
likelihood ratio chi-square test is significant.

Colle, J.G.; Duguid, J.P.; Fraser, A.G.; et al. (1996): Practical
Medical Microbiology, 14th edition. Edited by Mackie and McCartney.
Churchill, Livingstone.

 Del Pozo, J. (2008): Update and actual trends on bacterial infections
following liver transplantation. World J Gastroenterol ; 14(32): 4977–

García-Prado E, Cordero E, Alamo JM et al. (2009): Descriptive
study of infectious complications in 109 consecutive liver transplant
recipients Enferm Infecc Microbiol Clin. Apr;27(4):199-205.

Garner JS, Jarvis WR, Emori TG, et al.(1988): CDC definitions for
nosocomial infections, 1988. Am J Infect Control ; 16: 128.

Hashimoto M, Sugawara Y, Tamura S et al. (2008): Methicillin-
resistant Staphylococcus aureus infection after living-donor liver
transplantation in adults. Transpl Infect Dis. ;10(2):110-6.

Kanwal, F.; Dulai, G.S.; Spiegel, B.M.; et al. (2005): A comparison
of liver transplantation outcomes in the pre- vs. post-MELD eras.
Aliment. Pharmacol. Ther., 21(2):169-177.

Kawecki D, Chmura A, Pacholczyk M et al. (2009): Bacterial
infections in the early period after liver transplantation: Etiological
agents and their susceptibility. Med Sci Monit;15(12): CR628-637.

Moncorgé C, Baudin F, Vigouroux C, et al. (1989): Liver
transplantation in adults: postoperative management and development
during the first months. Ann Fr Anesth Reanim. ;8(5):497-517.

Morgan M, Madden A, Soulsby C et al. (2006): Derivation and
validation of a new global method for assessing nutritional status in
patients with cirrhosis.Hepatology ; 44:823-835.

Porrett PM, Hsu J and Shaked A. (2009): Late surgical
complications following liver transplantation. ;15 Suppl 2:S12-8.
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(3): 444-465.

Rubin RH (2002): The direct and indirect effects of infection in liver
transplantation: Pathogenesis, impact, and clinical management. Curr
Clin Top Infect Dis; 22:125-154.

Schneider C.R, Buell J.F, M. Gearhart M et al. (2005): Methicillin-
Resistant Infection in Liver Transplantation: A Matched Controlled
study. Transplantation Proceedings, 37(2):1243-1244.

Zhu, J.Y.; Gao, P.J.; Li, G.M.; et al. (2009): Analysis of 565 cases
of liver transplantation at a single transplantation center. Beijing Da
Xue Xue Bao, 41(3): 368-372.

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