Friday, July 29, 2011

Overview of Healthy Hospital Environment

By professor Dr Maysaa El Sayed Zaki
 In developed countries, less developed and developing countries, preventing hospital acquired infections (HAIs) is a mjor work for microbiologist. It represents every day challenge. Here we will give an overview of his short- long day work.
The risk of infection in hospitals is always present.

Patient may acquire infection before admission to the hospital = Community acquired infection.

Patient may get infected inside the hospital =Nosocomial infection.

It includes infections

Not present nor incubating at admission,

infections that appear more than 48 hours after admission,

Those acquired in the hospital but appear after discharge

Also occupational infections among staff Organisms causing N.I. can be transmitted to the community through discharged patients, staff and visitors.

If organisms are multiresistant they may cause significant disease in the community.

sThe microbial agent

-Many sick people are treated in a closed area; microorganisms, frequent contact between carriers & susceptible, contaminated waste, equipment and supplies to be handled.

-Developing of clinical disease depends on organisms virulence, infective dose and patient resistance Viruses: HIV, HBV, HCV can be also be

transmitted through blood & B F (transfusion, injections, dialysis)

respiratory syncytial virus, rota virus, ebola, infleunza, herpes simplex viruses.

Parasites & Fungi: e.g. Giardia lamblia is easily transmitted between adults or children, Aspergillus sp. Affecting imunocompromised.

Scabies an ectoparasite causing outbreak.

Patient susceptibility

Healthcare settings are environment where both infected persons and persons at high risk of infection congregate.

Ø Crowded conditions within hospital, frequent transfers of patients between units.

Ø Microbial flora may contaminate objects, devices and materials which subsequently contact susceptible body sites of patients.

Environmental factorsI.actors N.I.

NWhere do nosocomial infection come from?

üEndogenous infection: When normal patient flora change to pathogenic bacteria because of change of normal habitat, damage of skin and inappropriate antibiotic use. About 50% of N.I. Are caused by this way.

üExogenous crossinfection:

Mainly through hands of healthcare workers, visitors, patients.

several types of microorganisms survive well in the hospital environment (hospital flora):

* In water, damp areas and occasionally in sterile products or disinfectants eg pseudomonas, Acinetobacter, Mycobacterium.

* On items such as linen, equipment and supplies

* In food.

* In fine dust and droplet nuclei

Some procedures that save life may increase risk of infection e.g urinary catheters, I.V.L inhalation therapy, surgery.

Inappropriate use of antibiotics.

Prevention of nosocomial infection is the responsibility of all individuals and services provided by healthcare setting.

þ To practice good asepsis, one should always know: what is dirty, what is clean, what is sterile and keep them separate.

þ Hospital policies & procedures are applied to prevent spread of infection in hospital

Every Hospital should have a nosocomial infection prevention manual compiling recommended instructions and practices for patient care.

This manual should be developed and updated in a timely manner by the infection control team.

It is to be reviewed and accepted by infection control committee.

Aiming at preventing spread of infection:

Standard precautions: these measures must be applied during every patient care, during exposure to any potentially infected material or body fluids as blood and others.


A. Hand washing.

B. Barrier precautions.

C. Sharp disposal.

D. Handling of contaminated material.

1. Gloves:

Disposable gloves must be worn when:

a) Direct contact with B/BF is expected.

b) Examining a lacerated or non intact skin e.g wound dressing.

c) Examination of oropharynx, GIT, UIT and dental procedures.

d) Working directly with contaminated instruments or equipment.

e) HCW has skin cuts, lesions and dermatitis

-Sterile gloves are used for invasive procedures.

-GLOVES MUST BE of good quality, suitable size and material. Never reused.

2) Masks & Protective eye wear:

• MUST BE USED WHEN: engaged in procedures likely to generate droplets of B/BF or bone chips.

• During surgical operations to protect wound from staff breathings, …

• Masks must be of good quality, properly fixed on mouth and nasal openings.

3) Gowns/ Aprons:

Are required when: • Spraying or spattering of blood or body

fluids is anticipated e.g surgical procedures.

• Gowns must not permit blood or body fluids to pass through.

• Sterile linen or disposable ones are used for sterile procedures.

Needle stick and sharp injuries carry the risk of blood born infection e.g AIDS, HCV,HBV and others.

Sharp injuries must be reported and notified


Dispose of used needles and small sharps immediately in puncture resistant boxes (sharp boxes).

Sharp boxes: must be easily accessible, must not be overfilled, labeled or color coded.

Needle incinerators can be another safe way of disposal.

Reusable sharps must be handled with care avoiding direct handling during processing.

1. Cleaning of B/BF spills:

A- weargloves.

b-wipe up the spill with paper or towel.

c-apply disinfectant.

2. Cleaning & decontamination of equipment:

Protective barriers must be worn.

3. Handling & processing lab specimens:

must be in strong plastic bags with biohazard label

4. Handling and processing linen:

Soiled linen must be handled with barrier precautions, sent to laundry in coded bags.

5. Handling and processing infectious waste:

a. must be placed in color coded, leakage proof bags, collected with barrier precautions

b. contaminated waste incinerated or better autoclaved prior to disposal in a landfill.

Environmental control:

1. Including physical facility plans must meet quality of infection control measures.

2.Patient equipment positioning and installation, traffic flow.

3. Cleaning of hospital environment and disinfection according to policies.

• Cleaning staff has to be trained and instructed.

Surveillance at regular intervals has to be ensured.

• In risk areas with increased hazards (e. g. OR, intensive care therapy ward etc) special requirements have to be made for the deployment of cleaning staff which have to be defined in the infection control policy. Depending on size, risk areas and treatment frequency of each department

it can be necessary to assign special qualified personnel solely responsible for this particular area.

• Cleaning bucket and other containers for the storage of cleaning and disinfectant solutions and cleaning utensils have to be properly processed

following cleaning and disinfection procedures.

• The patient’s environment has to be clean, free of dust and soiling and should be in an optical acceptable and appealing condition for patient and

medical personnel.

• Cleaning and disinfection procedures have to lead to a reduction in the microbial count and to the death of pathogenic or facultative pathogenic


• By definition, cleaning procedures with detergents do not kill facultative pathogenic microorganisms.

They only remove soil and their killing effect on surface microorganisms is insufficient. Detergents can partly stabilize nonenveloped viruses and

promote the formation of spores, e. g. of Clostridium difficile.

• Furthermore, when solely using cleaning procedures without disinfectants this practice actually contributes to the spread of pathogens.

Therefore the aim must be to prevent contamination of the solution and the cleaning utensils by applying the appropriate procedures and products for cleaning and disinfection, to achieve the killing/inactivation of microorganisms in risk areas, to prevent the spread of pathogens and to ensure the interruption of potential infection chains.

• Cleaning and disinfection procedures must neither promote an increase in the microbial count nor a spread of facultative pathogenic microorganisms

(Pseudomonads, Enterobacteriaceae, Acinetobacter) on the treated surfaces.

Surfaces e.g. of medical appliances as well as all other horizontal surfaces should be relatively smooth, with sealed joints, without seams, washable and disinfectable with a disinfectant.

• Due to the difficulties in cleaning and disinfection of textile floor coverings, these should not be used wherever regular cleaning and disinfection is necessary for reasons of infection prevention.

• Any clothes used in cleaning floors have to be disinfected by washer machine at high temperature and the used stick should placed in active chlorine for 5minutes.

• The rooms for processing and storage of cleaning materials and utensils have to be sufficiently large and air conditioned. According to need appropriate washer disinfectors for the processing of cleaning utensils and

devices as well as drying appliances should be available.

• The surface to be disinfected has to be rubbed with slight pressure (wet mopping) using a sufficient amount of disinfectant.

• Open disinfection solutions should not be used for more than one day.

• In the event of severe contamination with organic material (blood, secretions, feces etc.) the visible material should be taken up with a cloth soaked in disinfection solution that has to be discarded afterwards. For this activity disposable gloves should be used. Subsequently the surface must be disinfected

Area without infection risk Examples Staircases, floors, Administration, offices, Dining areas, Lecture halls, Classrooms, technical areas:

The appropriate method is cleaning only.

• Areas with possible infection risk Examples General wards, Outpatient areas, Radiology, Physical therapy, Sanitary rooms, Dialysis, Accouchement, Intensive therapy/surveillance

• Surfaces with frequent hand / skin contact:

cleaning and Disinfection (most appropriate is cidex).

floors, doors, ceilings other surfaces:: cleaning

• Areas with special infection risks Examples OR departments, Intervention/OR rooms, Units for:

– special intensive therapy, e. g.: (patients receiving longterm

artificial respiration (> 24),

– transplantations (e. g. BMT, stem cells)

– haematooncology (e. g. patients under aggressive chemotherapy) premature infants

Surfaces with frequent hand/skin contact:




other surfaces:


• Areas with patients who carry pathogens in them or on them so that in individual cases there may be the risk of dissemination, TB, MRSA.

Surfaces with frequent hand/skin contact:




other surfaces:


• Areas in which there is an infection risk , particularly for personnel Examples Microbiology laboratories, Pathology, Disposal, Dirty areas

of: laundries function units, e. g. central sterilization

Cleaning and disinfection

• Control and Quality Assurance

• Disinfection and cleaning procedures and procedures for processing cleaning utensils have to undergo regular hygiene inspections, including the cleaning and disinfection of utensils and solutions.

By means of hygienic, microbiological examinations the efficacy of cleaning and disinfection procedures as well as the possible spread of facultative pathogenic microorganisms can be monitored.

3. Proper air ventilation.

4. Water pipes examination, check its quality.

5. Proper waste collection and disposal.

6. Cleaning and disinfection of equipment.

7. Proper linen collection, cleaning, Distribution

8. Food: ensure quality and safety.

9. Sterilization:

Central sterilization department serving all hospital departments compiling with infection control precautions.

Patient protection :

* corrective measures before major procedure, vaccination, proper use of antibiotics.

* Isolation precautions.

* Limiting endogenous risk

Staff health promotion and education:

1. HCW are at risk of acquiring infection, they can also transmit infection to patients and other employee.

2. Employee health history must be reviewed, immunizations recommendations to be considered.

3. Release from work if sick, occupation injury must be notified.

4. Continuous education to improve practice, better performance of new techniques.BB.I.

Thursday, July 28, 2011

Impact of Nosocomial infections

Hospital-acquired infections add to functional disability and emotional stress of the patient and may, in some cases, lead to disabling conditions that reduce

the quality of life. Nosocomial infections are also one of the leading causes of death (. The economic costs are considerable. The increased length of stay for infected patients is the greatest contributor to cost. One study showed that the overall increase in the duration of hospitalization for patients with surgical wound infections was 8.2 days, ranging from 3 days for gynaecology to 9.9 for general surgery and 19.8 for orthopaedic surgery. Prolonged stay not only increases direct costs to patients or payers but also indirect costs due to lost work. The increased use of drugs, the need for isolation, and the use of additional laboratory and other diagnostic studies also contribute to costs.

Hospital-acquired infections add to the imbalance between resource allocation for primary and secondary health care by diverting scarce funds to the management of potentially preventable conditions. The advancing age of patients admitted to health

care settings, the greater prevalence of chronic diseases among admitted patients, and the increased use of diagnostic and therapeutic procedures which affect the host defences will provide continuing pressure on nosocomial infections in the future.

Organisms causing nosocomial infections can be transmitted to the community through discharged patients, staff, and visitors. If organisms are multiresistant, they may cause significant disease in the community.

Wednesday, July 27, 2011

Mycobacterium Tuberculosis Clinical Aspects

Tuberculosis (TB) represents a major public health problem, especially in low-resource countries where the burden of the disease is more important. According to estimates of the World Health Organization (WHO), two billion people, roughly one third of the world’s population is latently infected with Mycobacterium tuberculosis the causative agent of the disease. In 2005 there were 8.9 million new cases of TB and 1.6 million deaths were attributed to the diseas. This scenario is aggravated by the human immunodeficiency virus (HIV) pandemic with roughly one third of the 40 million people currently infected with HIV also coinfected with TB.
The world can be divided into two parts based on the extent of tuberculosis epidemics. One part is the low-prevalence areas. They are composed of countries that experienced serious tuberculosis epidemics after the 18th century but have gradually overcome them and have finally reduced the incidence rate to 100 per 100 000 or less. The other part is the high-prevalence areas comprising countries with an incidence rate exceeding 100 per 100 000 that have suffered tuberculosis epidemics after the turn of the 20th century. The low-prevalence countries are industrialized countries, while the high-prevalence countries are mostly developing counties or areas. The latter accounts for two-thirds of the world population, but as much as 95% of the estimated number of newly occurring tuberculosis patients (of all forms) globally. Furthermore, 98% of tuberculosis deaths occur in these high-prevalence areas.1 Tuberculosis accounts for 2.7% of the total disability-adjusted life-years in low- and middle-income countries.4 In addition to the difference in its level, there are clear differences in characteristics of tuberculosis disease. In high-prevalence countries, most tuberculosis patients are in their 20s to 40s, resulting in tremendous socioeconomic loss as this is the most productive generation. In contrast, among low-prevalence countries, tuberculosis is drifting to involve the elderly, socioeconomically marginalized people, medical high-risk groups (e.g. diabetics5 and those treated with immunosuppressive agents, such as TNF-alpha blockers6), which presents a challenge to both medical and welfare services.
As a consequence of the global efforts in tuberculosis control under the Directly Observed Treatment Short-course strategy since the 1990s, the incidence of tuberculosis is estimated to have started to decline for the first time around 2003, although very slowly.1 At the same time, issues that had been given only lower priority in the developing world have emerged as unavoidable challenges. One of these issues is multi-drug resistant (MDR) tuberculosis that strikes a half million people annually and is a malignant burden to the patients and community, as well as to national tuberculosis programmes with its poor treatment outcome.7 In line with this problem, extensively drug resistant (XDR) strains of M. tuberculosis are emerging recently.8 The use of effective secondary drugs based on the result of high-performance drug sensitivity tests is necessary in order to address these issues, which requires technical innovation.7
A second newly emerging issue is co-infection of the HIV and M. tuberculosis. Currently, 15% of the new tuberculosis patients are infected with HIV, and in some areas or countries this proportion exceeds 50%. One quarter of the global tuberculosis deaths are due to HIV, and this is equal to one-third of new HIV-positive tuberculosis cases and to 23% of the estimated two million HIV-related deaths in 2007.1 Diagnosing tuberculosis in these subjects with sputum smear examination alone cannot prevent their infectiousness and save their lives; more aggressive case-finding and treatment of smear-negative cases are required. Another issue is tuberculosis in children for whom Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccination has been virtually the only control measure in developing countries. This also requires accurate diagnosis in the early stage of tuberculosis.9
As seen above, tuberculosis appears as a typical south-north problem of health, but currently in many developed countries over half of the new tuberculosis cases are foreign-born, that is, immigrants from high-prevalence areas, or spill-over of tuberculosis.10,11 It is actually argued that to further reduce tuberculosis in the low-prevalence countries it is necessary to strengthen control efforts in the high-prevalence, developing countries.12
Clinical Manifestation of Mycobacterium tuberculosis
In general, clinical manifestations of illness are the first clue to the diagnosis of tuberculosis. However, they are often non-specific and misleading, and therefore the diagnosis is not always easy. This is especially the case for tuberculosis in children and in elderly subjects, as seen below. Further, extrapulmonary tuberculosis often poses a challenge to early diagnosis, especially pertaining to its variety of presentations. Clinical signs and symptoms of tuberculosis of the organs other than the lungs are summarized in 134,302
Primary infection cases have generally an acute course of symptoms than reactivation-type TB. Cough (non-productive) and chest pain (pleuritic—sharp, stabbing, associated with respiration). Feverishness, dyspnea, chills, sweats, weight loss in more advanced cases. TB pleural effusions are generally exudates.
Most often in the neck and head region, rare in the axillary & inguinal region. Right predominates but 1/4 have biliateral & 78% have multiple lesions. 41% have pulmonary TB. In superficial LN, lesion starts as a painless enlargement, with no inflammation over the skin, then may undergo pustulation and fistulation over several weeks or months. In a case with limited lesion, general symptoms are rare
Bone & Joint
Commonest in vertebral column, followed by hip and knee. Fever and wasting may appear in large inflammatory collections, but the local manifestation predominates. Pain is commonest. Soft tissue collection (cold abscess) at/near the bone or joint focus. Neurological signs (weakness or numbness from compression of the spinal cord).
Disseminated or miliary TB
Diverse depending on the organs involved. Feverishness, weakness or debility, anorexia, weight loss, headache (meningeal complication), abdominal pain/swelling (peritoneal involvement), cough.39,40
Central nervous system
The presentation depends on the size and location of the tuberculoma and the pressure it produces.Early symptoms (feverishness, malaise, anorexia, irritability, headache) followed by neurological symptoms (progressive headache, lethargy, personality changes, memory disturbance, impaired cognition, confusion), and then stupor-coma with or without neurological deficit.
Cerebrospinal fluid for pressure, cellularity, protein, glucose,46 MTB (microscopy, culture47,48 and PCR49,50), immunology (ELISA, IgG immune complex, antibody assays and IGRA51–56), and ADA. Radiography, CT, MRI.57–61 Meningeal biopsy (histology, MTB).
Other infections (fungal, viral, trypanosomal, bacterial), vascular (multiple emboli, SBE, thrombosis of sagittal vein), collagen vascular (SLE, polyarteritis, and others).
Frequency: peritonitis, followed by ileocaecal, anorectal and mesenteric lymph node infection.In peritoneal TB, abdominal swelling, fever, ascites, pain, anorexia/weight loss are common.62,63
Peritonitis: ultrasound,64–66 laparoscopy (with guided biopsy),67–70 paracentesis of ascites for culture and IGRA71,72 and ADA.73
Malignant ascites, cirrhosis with spontaneous bacterial peritonitis, starch peritonitis, sarcoidosis, NTM peritonitis.
Dyspnoea, tachycardia, neck vein distension, oedema, hepatomegaly, paradoxical pulse, pericardial rub, fever.74,75
Pericardial tissue/fluid for bacteriology, histology,76,77 IGRA,78,79 and ADA.80–82 Echocardiography,83–85 CT and MRI (pericardial effusion and thickening),86 ECG (low voltage, inversion of T).87
Bacterial (e.g. Pneumococcus), viral (e.g. CMV, HSV, Coxsackievirus) or fungal (e.g. Aspergillus) infections; collagen vascular diseases; uremia; post-myocardial infarction or post-pericardiotomie; malignancy; trauma.
Dysuria, frequency, nocturia, urgency, pain in the back, flank or abdomen, tenderness/swelling of the testis or epididymis, haematuria. Superimposed urinary tract infection with other bacteria in urinary stasis cases.88–93
Tuberculosis in children
Because of its paucibacillary nature, tuberculosis of children is difficult to diagnose. Bacteriological confirmation seldom exceeds 30–40% among children in developed as well as developing countries.106,107 Consequently, the diagnosis of tuberculosis in children in resource-poor settings is largely dependent on a combination of a history of contact with a known tuberculosis patient, clinical signs and symptoms, and special examinations, such as chest radiography and the TST when available. Edwards and colleagues observed a total of 91 tuberculosis cases younger than 15 years, of whom about half were HIV-infected, and found the following frequency of symptoms and signs in the HIV-seronegative children: weight loss 69%, fever 100%, cough 83%, night sweat 43%, fatigue 21%, tuberculosis contact 60%, malnutrition 57%, lymphadenopathy 88%, organomegaly 31%, positive TST 89%, elevated erythrocyte sedimentation rate 79%, and chest X-ray infiltration 100%.108 Based on these observations, several point-scoring systems, diagnostic classifications and diagnostic algorithms have been developed to support an objective diagnostic judgment. Marais et al. tested such an approach and found that combining a persistent non-remitting cough lasting over 2 weeks, documented deterioration of health (in the preceding 3 months) and fatigue provided reasonable diagnostic accuracy in HIV-uninfected children (sensitivity 62.6%; specificity 89.8%; positive predictive value 83.6%). The performance was poorer in HIV-infected children than in the low-risk group, which offers a serious challenge in resource-poor settings with high HIV epidemics.109 However, given this set of sensitivity and specificity, the positive predictive value is calculated as only 24% in a patient population with a prevalence of tuberculosis of as high as 5%.
Tuberculosis in old ages
In low-prevalence situations, tuberculosis is a problem predominantly of the aged population and includes many more cases of clinical development in immunologically compromised subjects. This is why there are many tuberculosis cases with ‘atypical’ clinical presentation(s) in older persons.110,111 Elderly patients are more likely to have extrapulmonary tuberculosis, including miliary disease.111 The proportion of bacteriologically confirmed pulmonary tuberculosis patients was higher in the elderly than in the younger patients as reported in a meta-analysis.112
Fever, sweating and haemoptysis are less frequent in older patients, but dyspnoea is more frequent.112 Laboratory findings, such as the TST-positive rate, serum total protein level and white blood cell counts, were lower in elderly patients. Also, cavity formation was less common in elderly patients, while lesions in the upper lung were similar for both age groups.112 The most common chest X-ray findings in the elderly or immunocompromized tuberculosis patients are lesion in the lower zone accompanied by basal effusion or thickening.110 Such atypical clinical presentation of tuberculosis in the elderly can often cause delay in diagnosis, which can be further complicated due to underlying illnesses.
One of the basic indicators of quality in diagnosing tuberculosis is the delay in diagnosis (‘doctor's delay’, or ‘health system's delay’), that is, the time from the first visit of a patient until the establishment of tuberculosis diagnosis. Figure 2 depicts the delay separately for high-, intermediate- and low-prevalence settings, together with the patient's delay, that is, the time from the onset of clinical symptoms until the first visit to a health facility, based on published studies (T. Mori, pers. comm.). It is remarkable that these delays in the low-prevalence settings are always longer than those in the high-prevalence settings.
Sasaki et al. reviewed the diagnostic process of private practitioners with Japanese patients and concluded that insufficient medical work-ups, including AFB examinations of sputa and chest X-rays of subjects with a high suspicion for tuberculosis, was the principal cause of delayed diagnoses.113,114 In Hong Kong, general practitioners' practice was reviewed, and it became clear that they depend too much on X-rays rather than sputum examinations, and that they were slow in referring tuberculosis patients to the government tuberculosis service.115 Rozovsky-Weinberger et al. compared the management of suspect tuberculosis cases at three public hospitals and seven not-for-profit private hospitals in the USA in terms of their rates of ordering acid-fast smears and isolations, and urged private hospitals to be more alert to tuberculosis.116 Similar reviews of hospital management were reported by several other studies,117–119 the results of which illustrate the need for improved education of doctors. All of these studies urge a higher index of suspicion for tuberculosis in medical staff in low-prevalence countries.
need to read more
Read Mycobacterium tuberculosis: Current status in laboratory diagnosis

Hospital – acquired Infection

Definition :
       It is infection acquired while staying in hospital. It meets the following criteria:-
-                     Not found on admission.
-                     Temporally associated with admission or a procedure at a health care facility .
-                      It was not incubating at admission and may be related to a previous procedure or admission to same or other health care – facility e.g HIV acquired from previous blood transfusion .
Routes of infection: -
1)                Self infection (auto genous infection) .
2)                Cross – infection.
3)                Environmental infection
      -Bed linen
       -Air .
      -Moist solutions may be contaminated                         Pseudomonas                                                                                                                       
Factors which promote hospital acquired infection: -
1)                Impaired general host defenses of the patient.
2)                Impaired local host defenses of the patient .e.g injury of skin barrier.
3)                Presence of hospital pathogen which are endemic.
Organisms causing Hospital acquired infection: -
In many hospital but also each hospital has specific endemic or epidemic strains of particular types of organism in certain areas .
In hospitals with large specific units e.g oncology , special care baby units opportunists organisms as well as the common pathogens are likely to cause problem
 Pathogens causing hospital acquired infections include
1)    Conventional pathogen e.g strepto-pyogenes .
2)    Conditional pathogen e.g Bacteroides .
3)    Opportunists pathogen e.g Pneumocystis carinii .
The most common organisms are staph. aureus and staph.epidermidis
-Gram Negative bacilli such as: -
 E.coli is the most frequent single bacterial species associated with hospital acquired infection .
Klebsiella , Proteus species and Pseudomonas are also common cause .
- Fungal and viral infection are only occasionally acquired in hospital .
- Protozoa are rare.
Laboratory diagnosis Of Hospital – acquired Infections :
-Samples collected from the patient includes Blood culture, sputum, tracheostomy wound swabs and other samples. Perform cultured before antibiotics therapies and judged accurately if the isolate is just contamination or already a true pathogen.
-Samples from the environment include all equipments in the ICU and from antiseptic solutions according to the place of environmental assay.
-Typing of the isolated organisms from both patients, environment and personnel by the following typing methods:
1)                Bio typing
2)                Sero typing
3)                Phage typing
4)                Bacteriocine typing
5)                DNA finger printing
2-Surgical wound Infection
1) The type of the wound is the most single factor associated with the development of wound infection . The major types of surgical wounds can be classified as follows : -
-Clean operation wounds in area not involve regions of gastrointestinal tract, respiratory tract or genitourinary tract. It is associated with very low rates of infection 2-5%.
- Contaminated operation Surgery that involves a site with known normal flora (apart from skin) e.g. operation on colon , gall bladder , mouth or vagina .
- Infected operation wounds , the operation site may be infected at time of surgery e.g. incision of an abscess .
2) Surgical team:
- Skill of surgeon
- Good aseptic techniques
- Carriage of staph. aureus .
3) Age and general condition of the patient .
4)    Persistence of local structural abnormal .
5)    Ward factors post operatively
Complication of wound Infections : -
-                     Delayed wound healing
-                     Failure of graft .
-                     Infections of bones , joints , peritoneal cavity .
-                     Septicemia
Types of hospital acquired wound infections include : -
-                     Ward infection
-                     Theatre infection
(How to differentiate ?)
Prevention of theatre infection : -
-                     Protective of  theatre clothing .
-                     Gloves for the hands after chlorhexidine antiseptic solution
-                     Movements of staff should be reduced to a minimum .
-                     Very clean theatre with good managing of the air direction around the operative table .
-                     Disinfect the skin of the patient and many use preoperative baths with hexachlorophene or chlorhexidine detergents .
Prevention of wards infections : -
-                     Isolation rooms for wards should be available for patients infected with MRSA or with severe wound infections especially in high risk surgery e.g. cardio thoracic orthopedic , neuro surgical units and ICU .
-                     Adequate non touch technique for dressing of the wound .
-                     Suitable bad spacing between patients (2.5m)to decrease air or dust spread .
-                     The patient should be admitted for the shortest time before the operation to decrease the chances of colonization or infection with hospital strains .
-                     Restrict the use of prophylactic antibiotics .
-                     Exclude patients with skin disease from the word .
-                     Exclude members of staff with boils , abscess or other infected skin lesions .
If an out breaks occur , how to control ?
-                     Be sure that it is cause by the same epidemic strain with similar antibiotic resistance pattern and phage type .
-                     Isolate the patient .
-                     The medical staff shown to be carrier should temporarily cease the work and use chlorhexidine on the affected area with all hygiene measure .
-                     Close the ward in severe cases .
-                     Use vancomycin for therapy .
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Sepsis in Children with Oncohematological Malignancies, old Problem Revisited

   Though the use of aggressive treatment protocols that combine chemotherapy, radiation, and surgery, the prognosis for patients with childhood malignancies has improved substantially. However, these intensive treatment regimens can cause life-threatening complications, the
most prominent of which are infections that result from treatment-associated immunosuppression.
Historically, sepsis and septic shock in pediatric oncology patients have carried a poor prognosis. Earlier studies, however, used a variety of criteria to define sepsis and septic shock, and most included relatively small numbers of children. Some studies even excluded patients who had received bone marrow transplants (BMT).
Sepsis is a leading cause of death in critically ill patients despite the use of modern antibiotics and resuscitation therapies. The septic response is an extremely complex chain of events involving inflammatory and anti-inflammatory processes, humoral and cellular reactions and circulatory abnormalities. The diagnosis of sepsis and evaluation of its severity is complicated by the highly variable and non-specific nature of the signs and symptoms of sepsis. However, the early diagnosis and stratification of the severity of sepsis is very important, increasing the possibility of starting timely and specific treatment.
    A rapid microbiological diagnosis could therefore confirm an infectious cause of fever and aid in the choice of a specific therapy. Among the infectious causes, bacteria and fungi are the leading threats, with high infection-related mortality rate, especially for polymicrobial infections and moulds. The current gold standard for the detection of bacterial pathogens in blood is blood culture. However, all blood culture systems suffer from several limitations, such as lack of rapidity and low sensitivity, especially when the patient has already received antibiotics and when fastidious micro-organisms are involved. From this perspective, the diagnosis of bloodstream infections could prove really challenging in oncohaematological patients, who routinely receive prophylactic antibiotics and whose blood cultures therefore often remain negative. Even after the detection of growth in cultured blood (usually not before 6–12 h of incubation), conventional blood cultures require at least a further 24–48 h for the definitive identification of the pathogen and the evaluation of its sensitivity to antibiotics. Other parallel approaches are therefore needed, and among them well-designed molecular assays could prove really useful. Several molecular techniques have already been successfully used in routine microbiology laboratories for direct detection of viral, bacterial, mycotic and protozoan pathogens. However, their use on whole blood samples for detection of sepsis has been hampered by several factors, including insufficient sensitivity, presence of PCR inhibitors in blood, and the difficulty of setting up an assay capable of detecting a wide range of potential pathogens.
  Another advance in diagnosis of sepsis is the use of biomarkers. Biomarkers can have an important place in this process because they can indicate the presence or absence or severity of sepsis. A biomarker is defined as ‘‘a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.’’.
 Beside rapid diagnosis of sepsis, other potential uses of biomarkers include roles in prognostication, guiding antibiotic therapy, evaluating the response to therapy and recovery from sepsis, differentiating Gram-positive from Gram-negative microorganisms as the cause of sepsis, predicting sepsis complications and the development of organ dysfunction (heart, kidneys, liver or multiple organ dysfunction). However, the exact role of biomarkers in the management of septic patients remains undefined. C-reactive protein (CRP) has been used for many years but its specificity has been challenged. Procalcitonin (PCT) has been proposed as a more specific and better prognostic marker than CRP, although its value has also been challenged. It remains difficult to differentiate sepsis from other non-infectious causes of systemic inflammatory response syndrome, and there is a continuous search for better biomarkers of sepsis.
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Sunday, July 24, 2011

Lectures in Clinical Microbiology: Classification & pathogenicity of Microbes

Lectures in Clinical Microbiology: Classification & pathogenicity of Microbes

. Laboratory Diagnosis of Hepatitis C Virus Infection.

Due to the lack of typical symptoms diagnosis of acute hepatitis C is rarely established. Because HCV antibodies may develop later during the course of hepatitis C diagnosis of acute infection is based on the detection of HCV-RNA together with a history of contact to a HCV positive source, a course of increased liver enzymes from previous normal levels and/or the absence of HCV antibodies (Jaeckel et al.,  2001 and Gerlach et al., 2003). 
            After screening for chronic HCV infection by anti HCV antibody testing past or ongoing hepatitis C in anti-HCV positive patients is determined on the basis of HCV core antigen and HCV-RNA. During selection of patients for screening of hepatitis C one has to be aware that typical symptoms of chronic liver damage and even elevated liver enzymes are often not present in patients with HCV infection. This together with a relative slow progression of the disease has led to the fact that at present only in a minority of patients diagnosis of  CHC is established.  After proof of CHC the need for antiviral therapy has to be assigned on the basis of the level of liver enzymes, histological grading and staging of liver damage, extra hepatic manifestations of the disease, as well as the social and personal situation of the patient (National  Institutes of  Health, 2002).
             For management of treatment, determination of HCV genotype provides important information about the chance of sustained virologic response and the projected duration of interferon alfa-based antiviral therapy (Hadziyannis et al., 2004 and Zeuzem et al., 2004).
             The key parameter for assessment of antiviral response during therapy is HCV-RNA. Precise and reliable quantification and highly sensitive detection of HCV-RNA before, during and after interferon alfa based antiviral therapy is critical for determination of virologic response and premature discontinuation of therapy in virologic-non-responders (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003 and Davis et al., 2003). Detection of HCV antibodies.
            The first enzyme immunoassay was introduced in 1989/90 and was able to detect antibodies to a c100–3 named antigen, a recombinant protein from the non-structural (NS)4 region of the HCV genome (Ortho Diagnostic Systems Inc., Raritan, NJ, USA) (Kuo et al., 1989).
            Today’s third-generation enzyme immunoassays (EIA-3) are available since 1993 and were completed with recombinant proteins of the NS5 region. In comparison with the second generation assays (detection of core, NS3 and NS4 proteins) a 2–3 weeks earlier detection of HCV antibodies was shown and specificity to detect HCV antibodies in blood donors was improved from 99.90% to 99.95% (Uyttendaele et al., 1994 and Barrera et al., 1995). Sensitivities in patients with chronic liver disease and in panels of sera were estimated at 98.90% and 97.20%, respectively (Colin et al., 2001). Generally, results for anti-HCV screening are only reported as positive or negative. Differentiation of acute and chronic hepatitis C may be possible by quantification of anti HCV core IgG levels. IgG antibodies levels typically are higher in chronic compared to acute hepatitis C and usually persist in immunocompetent patients (Nikolaeva et al., 2002).  After spontaneous recovery from acute hepatitis C or successful eradication of the virus, disappearance of anti-HCV IgG below the detection limit of the assays may be seen after many years (Wiese et al., 2000). Detection of HCV antigens.
            After screening for HCV infection with HCV antibody tests confirmation of ongoing hepatitis C is performed by the detection of HCV core antigen and HCV-RNA. For routine purposes HCV antigens will be detected in the blood while for scientific research liver tissue or lymphocytes may be used alternatively. After acute infection HCV-RNA will become detectable within the first 2 weeks and during chronic infection virtually all patients are HCV-RNA positive using sensitive assays. Due to a lower sensitivity the use of HCV core antigen tests instead of HCV-RNA assays with some restrictions will lead to the same results. During chronic hepatitis C, HCV core antigen and HCV-RNA assays are used for monitoring of antiviral therapy for prediction and confirmation of virologic response, before, during and after treatment (Bouvier-Alias et al., 2002 and Veillon et al., 2003). HCV core antigen.
            The introduction of a reliable and sensitive HCV core antigen assay was hampered by different difficulties: (i) the development of specific monoclonal antibodies recognizing all different HCV subtypes; and (ii) the need of accumulation and dissociation of HCV particles from immune complexes for increasing sensitivity. Recently, a first HCV antigen detection system was approved by the legal authorities and has become commercially available in the US and Europe (Ortho, trak-CTM, Ortho Clinical Diagnostics, Raritan, NJ, USA). In this assay, after dissociation of HCV particles from immune complexes and lysis, HCV core proteins are bound to coated monoclonal antibodies in a microwell. Following several washing steps a bound core antigen is incubated with an anticore-specific Fab antibody fragment conjugated with horseradish peroxidase. After a second wash quantitative detection is performed by the addition of ophenylenediamine (OPD)/hydrogen peroxide and measurement of the optical density. HCV core concentrations are calculated against a curve obtained from standards and are expressed as picograms per milliliter.  The limit of detection established by the manufacturer is 1.5 pg/mL. The HCV core antigen assay is highly specific (99.5%), genotype independent, and has shown a low inter- and intra-assay variability (coefficient of variation 5–9%) (Bouvier-Alias et al., 2002 and Veillon et al., 2003).
            After infection with HCV detection of HCV core antigen is delayed approx. 1–2 days after HCV-RNA becomes detectable (Icardi et al., 2001 and Cividini et al., 2003).
            As for HCV-RNA, no correlation between levels of HCV core antigen and elevation of liver enzymes or the grade of inflammation and the stage of fibrosis in the liver was observed. From the results of different studies it was shown that the lower detection limit of 1.5 pg/mL for core antigen equals to approximately 10,000–50,000 IU/ml (Bouvier-Alias et al., 2002 and Veillon et al., 2003). 
            In a study with 139 HCV antibody and HCV-RNA positive patients presented in an outpatient clinic six (4%) were HCV core antigen negative.  In these patients HCV-RNA concentrations were measured between 1,300 and 58,000 IU/mL highlighting the limitations to use the HCV core antigen assay for confirmation of ongoing hepatitis C in HCV antibody positive patients (Veillon et al., 2003).
            Comparison of HCV core antigen and HCV-RNA quantification show an excellent correlation (correlation coefficient, r =0.92). One pg/mL of HCV core antigen equals to 8,000 IU/mL HCV-RNA with a intersubject variation from 5,000 to 12,000 IU/mL (Bouvier-Alias et al., 2002 and Veillon et al., 2003)  Studies for evaluation of the utility of HCV core antigen concentrations for prediction of virologic response/non-response before and during interferon-based antiviral therapy are currently under way. Preliminary data of a small study investigating 38 patients treated with peginterferon-alfa and ribavirin observed a potential use of a positive HCV core antigen test (>1.5 pg/mL) for discontinuation at week 12 with a high negative prediction value (100%non-response) (Buti et al., 2004). 
            However, as shown by others, due to the limited sensitivity transient negative results by the HCV core antigen assay were associated with continuously positive HCV-RNA concentrations. The aim of determination of early virologic response certainly is to discontinue as many virologic non-responders as possible from further treatment with an ideal 100% prediction value. Thus, the proportion of nonresponder patients to be discontinued from further treatment at week 12 and 24 by core antigen and HCV-RNA measurement, respectively, has to be carefully assessed in large cohorts of patients (Rebucci et al., 2003). Detection of HCV-RNA. Qualitative assays (RT-PCR, TMA).
            After detection of HCV in 1989 the first test systems to confirm ongoing, replicating hepatitis C were based on RT-PCR. Due to a high conservation of the 5/ nontranslated region (NTR) of the HCV genome throughout the different geno- and subtypes, primers complementary to this region were chosen for reliable diagnostics (Christoph, 2004).
             Due to their lower detection limits in comparison with quantitative HCV-RNA assays, qualitative HCV-RNA tests clinically are used for diagnosis of acute hepatitis C in which HCV-RNA concentrations are fluctuating and may be very low and for confirmation of virologic response during, at the end and after antiviral therapy (Christoph, 2004). 
            By the end of 1993 a first standardized RT-PCR based assay for detection of HCV-RNA was introduced (AmplicorTM HCV, Roche Molecular Systems, Pleasanton, CA, USA). The Amplicor™ HCV system is a combined single tube-, single enzyme-, single primer set RT-PCR assay (Christoph, 2004). 
            More recently, a second test system based on transcription mediated amplification (TMA) was approved for qualitative detection of HCV-RNA (VersantTM Qualitative HCV, Bayer Diagnostic, Emeryville, CA, USA). HCV-RNA detection by this technique consists of threesteps which are performed in a single tube: (i) target capture; (ii) target amplification and; (iii) specific detectionof target amplicons by hybridization protection assay.
            Due to its extreme high sensitivity the TMA-based assay (lower detection 5–10 IU/mL) is able to detectresidual HCV-RNA amounts not observed by standard RT-PCR-based tests (lower detection limit 50 IU/mL). In 7–33% of patients treated with (pegylated) interferon-alfa and ribavirin with negative results by RT-PCR at the end of treatment residual HCV-RNA  concentrations were detectable by TMA (Sarrazin  et al., 2000; Comanor et al., 2001 and Sarrazin  et al., 2001).
            Similar results were obtained during therapy at week 24 for decisions of treatment discontinuation in HCV-RNA positive patients. Whether patients with low HCV-RNA levels (TMA positive/RT-PCR negative) will benefit from prolongation of therapy is still unknown (Mihm et al., 2004). Quantitative assays (RT-PCR, real time RT-PCR bDNA)
            Measurement of HCV-RNA concentration in serum/blood is an important parameter for management of chronic hepatitis C. While no correlation was observed between the HCV-RNA viral load and the severity or the progression of liver disease HCV-RNA concentration is an important predictor for response to antiviral therapy. In multiple studies an inverse correlation of lower pretreatment HCV-RNA blood levels with higher rates of sustained virologic response to (pegylated) interferon alfa combination therapy with ribavirin was observed. Furthermore, after initiating interferon-based therapy the initial decline of HCV-RNA levels is used for determination of virologic non-response at week 12 and week 24 of treatment. Patients without an initial decline of HCV-RNA of at least 2 log steps and/or absolute values above 30,000 IU/mL have shown to become virologic non-responders in 98–100% of cases (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            Thus, according to current recommendations treatment should be discontinued on the basis of this week 12 HCV-RNA decline rule. Highly sensitive HCV-RNA detection assays (lower detection limits ≤50 IU/mL) are used for determination of virologic response at week 24 of therapy. As patients with detectable HCV-RNA at this time point will not achieve a sustained virologic response in 98–100% of cases again treatment can be discontinued at week 24 on the basis of a positive highly sensitive HCV- RNA test (Fig. 4) (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
Figure 4 . Management of (pegylated) interferon alfa/ribavirin combination therapy (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            Figure (5) gives an overview on commercially available qualitative and quantitative HCV-RNA detection assays. Different techniques are used for quantification of HCV-RNA: (i) assays with HCV-RNA (target) amplification on the basis of standard or real time PCR; and (ii) a branched DNA (signal) amplification method without multiplication of the original amount of HCV-RNA in the probe (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            Five different quantitative HCV-RNA detection assays are approved by the legal authorities and are commercially available. Three are based on standard RT-PCR (SuperquantTM, National Genetics Institute, Los Angeles, CA, USA; Cobas AmplicorTM HCV Monitor version 2, Roche Molecular Systems, Pleasanton, CA, USA; LCxTM HCV-RNA Quantitative, Abbott Laboratories, North Chicago, IL, USA) and one is based on branched DNA technique (VersantTM Quantitative HCV-RNA, bDNA 3.0, Bayer Diagnostics, Emeryville, CA, USA). Recently, the first real-time PCR based assay for HCV-RNA quantification was approved (COBAS TaqManTM , Roche Molecular Systems, Pleasanton, CA, USA) and in the future additional real-time PCR based HCV quantification assays may follow as this technique has the potential of highly sensitive, linear quantification of RNA and DNA targets (Cristoph, 2004).

Figure 5. Characteristics of qualitative and quantitative HCV RNA detection assays. Numbers below the balls/columns represent lower detection limits of the assays. (●) qualitative assays; (■) real time PCR-based linear quantitative assays; (▓) standard PCR-based non-linear quantitative assays; (░ ) signal amplification bDNA-based linear quantitative assay (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
            HCV-RNA extraction from blood samples before detection with the HCV-RNA quantification assays is performed by different techniques (i.e. standard lysis and precipitation, glass fiber filter columns, hybridization to magnetic particles). To yield lower detection limits between 10 and 50 IU/mL higher sample volumes are needed for extraction of HCV-RNA. While for the real-time PCR-based Cobas TaqManTM assay as well as for the TMA-based assay 500mL are used for HCV RNA preparation, nucleic acids are extracted from 100mL and 50mL in the Cobas AmplicorTM Monitor and bDNA 3.0 assays, respectively. Automation of nucleic acid extraction requires a further increase with samples volumes up to 1 mL (Christoph, 2004).
            Lower and upper detection limits of linear amplification of HCV-RNA together with intra and interassay variabilities of the different assays are summarized in (Table 1). Taken together, precision data of the different standard PCR assays are comparable. The signal amplification based bDNA assay showed a slightly lower variability. The slightly higher variability of the real-time PCR based Cobas TaqManTM assay is explained by the extreme wide range of linear amplification from 35 to 7 Mill. IU/mL. Together with HCV-RNA preparation by the glass fiber filter-based HPSTM system the Cobas TaqMan assay at present is restricted for the use of quantification of HCV genotypes 1 and 6. For the remaining HCV genotypes (2, 3, 4, 5) underestimation of HCV-RNA concentrations were observed. The reason for this underestimation so far is unknown and studies to resolve the problem are currently under the way(Yu et al., 1999; Ross et al., 2002; Leckie et al., 2004 and Sarrazin et al., 2004).
            All commercially available HCV-RNA quantitative assays are standardized against the WHO HCV international standard (96/790) and titer results are automatically reported in international units (IU/mL). For calculation of results of previous versions of the different assays given in copies individual conversion factors to IU exist (Table 2) (Pawlotsky et al., 2000). While during long-term storage a significant decrease of HCV-RNA levels may be observed (Schmid et al., 1999), storage at room temperature during the first 72–96 h after blood collection was not associated with a decline of HCV-RNA concentrations (Grant  et al., 2000 and Kessler et al., 2001).

HCV RNA quantification assay



mean SD

mean SD

Amplicor MonitorTM HCV, version 2.0


5 × 105

0.080 log10

0.063 log10

LCxTM HCV Quantitative


2.3 ×106

0.075 log10

0.066 log10

SuperquantTM HCV





Cobas TaqManTM HCV


7 × 106

0.088 log10

0.103 log10

VersantTM Quantitative HCV, version 3.0


8 × 106

0.050 log10

0.030 log10

Table 1. Characteristics of commercial HCV-RNA quantitative assays *Data obtained from (Yu et al., 1999; Ross et al., 2002; Leckie et al., 2004 and Sarrazin et al., 2004). LDL, lower detection limit; UDL, upper detection limit; Inter-assay, inter-assay variability; Intra-assay, intra-assay variability; n.a., data not available.
Table 2. Conversion factors from copies to IU (Pawlotsky et al., 2000).

HCV RNA quantification assay

Conversion factor

Amplicor Monitor HCV, version 2.0

1 IU = 2.5 copies/mL

LCx HCV Quantitative

1 IU = 4.3 copies/mL

Superquant HCV

1 IU = 3.4 copies/mL

Versant Quantitative HCV,version 3.0

1 IU = 5.2 copies/mL

2.2.11. Prevention.
            The development of an effective HCV vaccines remains an unsolved challenge. This is due to the lack of suitable cell culture system and small animal models to establish HCV infection for evaluating crucial virus neutralizing pathways. During the last decad, many efforts have been dedicated to the design of either prophylactic or therapeutic HCV vaccines, but still with little success (Forns et al., 2002).
            Passive protection using high-titer specific immunoglobulin is also currently unavariable. At present the role of specific HCV neutralizing antibodies in the prevention and control of infection has not been defiend clearly, prevention of HCV transmission can be achieved only by preventing exposure to the virus. HCV transmission can be minimized by educating intravenous drug users not to reuse or share syringes and needles or any other items involved in drug taking, simple techniques for sterilizing such equipment have been suggested for preventing HCV transmission in intravenous drug users. Organ donors should be screened for evidence of HCV infection before transplantation, also transmission through sexual contact can be minimized by protected sex. Many different approaches have been currently tested using structural (E1-E2-Core) and non structural HCV protein as candidates for different strategies of immunization (Choo et al., 1994; Geissler et al., 1997 and Forns et al., 2002). New adjuvants are also evaluated to improve immunogenicity and to favor the generation of a strong cellular response (Sfolander and Cox, 1998).