Infection control history

Even before the birth of the germ theory, the courageous people who cared for the sick tried as hard as they could to protect themselves from contagion. In the late middle Ages, for example, the brave souls who volunteered to care for victims of the plague wore masks designed to shield them from the vapors (or miasmas) that they thought transmitted this lethal illness. Modern concepts of disease transmission are more sophisticated, and personal protective gear has evolved accordingly. In the industrialized world, high-performance gloves, waterimpermeable gowns, highly efficient masks, and impervious face shields are all part of the contemporary health care worker s standard gear.

Introduction to nosocomial infection and nosocomial Outbreaks

The National Nosocomial Infections Surveillance System (NNIS) system). The NNIS system de nes a nosocomial infection as a localized or systemic condition 1) that results from adverse reaction to the presence of an infectious agent(s) or its toxin(s) and 2) that was not present or incubating at the time of admission to the hospital. For most bacterial nosocomial infections, this means that the infection usually becomes evident 48 hours (i.e., the typical incubation period) or more after admission. However, because the incubation period varies with the type of pathogen and to some extent with the patient s underlying condition, each infection must be assessed individually for evidence that links it to the hospitalization

 They may be endogenous, arising from an infectious agent present within a patient s body, or exogenous, transmitted from another source within the hospital. In addition to patient-topatient spread, others may be involved, including staff, students, visitors, and voluntary workers. Infections that are in the incubation period at the time of admission to hospital are not classed as nosocomial, but community-acquired infection of patients or staff can be an important source of nosocomial infection

 An outbreak may be defined as a relatively rapid increase in the incidence of infection or colonization by a certain bacterial strain, caused by enhanced patient-to-patient transmission (van Belkum et al., 2007). Hospital-acquired outbreaks account for approximately 2-10% of all hospital-acquired infections. Just like endemic hospital-acquired infections, infections in outbreaks contribute signi cantly to morbidity and mortality,

 Socio-economic impacts of nosocomial infection (NI), including prolongation of hospital stay, increment of medical costs and deaths, are well recognized. However, most studies focusing on fatality among patients with NI have been discrete and specific in nature (e.g. elderly patients; stroke victims; patients in intensive care units [ICUs]; specific body sites including blood, respiratory tract or surgical wound; and infections with multidrug resistant micro-organisms including methicillin-resistant Staphylococcus aureus, extended-spectrum -lactamase producing Enterobacteriaceae, and Pseudomonas aeruginosa).

 Deaths were considered to be associated with NI if the NI was still active and being treated with antibiotics when death occurred and no other obvious causes of death were evident. NI was diagnosed according to the definitions of the United States Centers for Disease Control and Prevention. Severity of underlying disease classification was evaluated at the time of NI occurrence, using an established modified risk stratification regimen, as rapidly fatal (death anticipated during hospitalization), ultimately fatal (death anticipated within 5 years), or non-fatal (disease unlikely to be fatal within 5 years or absence of underlying disease)

Gram positive cocci Hospital-Associated MethicillinResistant Staphylococcus aureus (MRSA):

Infection control measures have been put into place in many hospitals with varying success however; studies have shown that even with strict hand-washing and contact isolation guidelines, MRSA is still capable of spreading (Boyce et al., 2004). For instance, even after proper hand washing and glove usage, MRSA could still be found on the hands of health-care workers (McBryde et al., 2004). Lack of education and poor compliance of the health-care workers might be contributing factors.

Source Tracing (Typing)

Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Nucleic acid techniques, such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into clinical laboratories. PCRbased systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and userfriendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques

Criteria to Evaluate Typing Systems

At this time, no typing system completely ful lls all the criteria described below, and the advantages and disadvantages of a technique should be understood before use: 1. Typeability: an unambiguous result for each isolate analyzed. Nontypeable isolates have a null or uninterpretable result. 2. Reproducibility: the same result when the same strain is tested repeatedly. Reproducibility is in uenced by technical factors (day-to-day variation) and biological factors (variation in the stability of the typing characteristic). 3. Discriminatory power: differentiates between unrelated strains but varies as species vary in genetic stability under the in uence of selective pressure. 4. Ease of interpretation: multiple users of the typing system obtain the same results and reach the same conclusions. Ideally, guidelines exist for interpretation. 5. Ease of performance: the rapidity, convenience, cost of equipment, and personnel needed to perform a technique

Molecular typing methods Advantages of molecular typing over other typing methods
The difficulty in assigning an organism to a biologically meaningful category should be well considered before the use of any molecular identification tool. Researchers should be aware of the evolutionary history and taxonomic position of the specimen under study. Ideally, they should understand the order of branching and ages of divergence (phylogeny) of the organisms in examination and be familiarized with the nomenclature used in previous studies. Terms such as strain , variant , subspecies or breed could be highly subjective in some circumstances and be used as synonyms by different investigators to describe the same biological entity

 However, with the enormous in uence of molecular biology in the 1970s it became increasingly clear that phenotypic characterization was only an expression of the organism s underlying genotype, several levels removed from the true genetic identity of the organism. Thus, molecular methods began to nd application in clinically relevant areas including epidemiological analysis, giving

 this transition can be viewed as a multi-step process the rst iteration of which was analysis of isolate plasmid content by agarose-gel electrophoresis. However, the bacterial genome is the most fundamental molecule of identity in the cell and thus represents the ultimate metric for assessing isolate interrelatedness. This principal is the foundation for second-generation (restriction enzymes and probes), third-generation (pulsed eld gel electrophoresis [PFGE] and PCR), and fourthgeneration (DNA sequence) molecular approaches to epidemiological analysis

 it should be realized that a continuous genetic variability does exist among individuals under study has to be properly assessed before undertaking any taxonomic identification in order to guarantee that there is no overlap between intraspecies variation and interspecies divergence

 If a new method or genetic marker will be employed for the first time, the genetic composition of all species of that taxon should be determined. If possible, a representative sample of individuals should be genotyped, preferentially from different geographic locations (or from different hosts, in cases of internal parasites). The preservation of voucher specimens to serve as a future reference is also highly recommended.

 It is also important to keep in mind that there is no perfect DNA-typing method and that the choice of a particular technique is often a compromise that depends on a number of factors, including: the resources of the laboratory, financial constraints, available expertise, time limitations and, more importantly, the research question pursued. All points should be scrutinized carefully to avoid an inappropriate choice of a species

Performance Comparison of Representative Molecular Epidemiology Methods

Method Typing Capacity
Good

Discriminatory Power

Reproducibility

Ease of Use

Ease of Interpretation

Plasmid analysis

Good

Good

High

Good

PFGE Chromosomal RFLP

High High

High Good

High Good

Moderate High

Good moderate Moderate poor

Ribotyping PCR-RFLP RAPD AFLP Repetitive elements

High Good High High Good

High Moderate High High Good

High Good Poor Good High

Good High High Moderate High

High High Good-High High High

Sequencing

High

High

High

Moderate

Good-High

Fig. (1). Schematic representation of most important procedural steps involved in the various DNA-based methods (Pereira et al., 2008).

Sample collection, preparation and quality control Specimen Collection: Although viability is not as critical for molecular testing, the quality of nucleic acids may be compromised if the specimen is improperly handled. DNA and especially RNA can be damaged in lysed or nonviable cells. Due to the sensitivity of molecular testing, it is also important to avoid contamination that could yield false-positive results

 Sampling must include material from the original infection. The time and site of collection must be optimal for the likely presence of the infectious agent. For example, Salmonella typhi is initially present in peripheral blood but not in urine or stool until at least 2 weeks after infection. For classical methods that include culturing of the agent, a sufficient number of microorganisms (103/ml specimen) must be obtained for agar or liquid culture growth. For molecular testing, however, minimum numbers (as few as 50 organisms) can be detected successfully. The quantity of target organisms as well as the clinical implications should be taken into account when interpreting the signi cance of positive results, as molecular detection can reveal infective agents at levels below clinical signi cance.

Sample Preparation:
First, depending on the microorganism, more rigorous lysis procedures may be required. Mycobacteria and fungi in particular have thick cell walls that are more difficult to lyse than those of other bacteria and parasites. Gram-positive bacteria having a thicker cell wall than gramnegative bacteria may require more rigorous cell lysis conditions. Mycoplasma, on the other hand, lacks a cell wall, and so care must be taken with the sample to avoid spontaneous lysis of the cells and loss of nucleic acids.

 Second, the concentration of organisms within the clinical sample must be considered. Samples should be centrifuged to concentrate the uid and the

 Third, inhibitors of enzymes used in molecular analysis may be present in clinical specimens; removal or inactivation of inhibitors must be a part of specimen preparation. Finally, if RNA is to be analyzed, inactivation or removal of RNases in the sample and in all reagents and materials that come into contact with the sample must occur

Quality Control:
Thus, ensuring that the integrity of specimens is maintained, i.e., that specimens are not contaminated by other specimens or with the products of previous ampli cation procedures, is critical to avoid false positives. On the other hand, it is equally important to ensure that the lack of a product in an ampli cation procedure is due to the absence of the target organism and not the presence of inhibitors preventing the ampli cation of target sequences (false negative)

PCR-Based Strain Typing Techniques

First, DNA is an extremely stable and long-lived biological molecule that can be recovered from biological material that has been under stress conditions (processed food products, coprolites, mummified plant tissues, blood stains, etc.). Second, DNA is found in all biological tissues or fluids with nucleated cells (or non-nucleated cells with plastids and/or mitochondria), enabling its analysis from almost all kinds of biological substrates (saliva, faeces, plant seeds, milk, etc.). Finally, DNA can provide more information than proteins due to the degeneracy of the genetic code and the presence of large non-coding stretches