key: cord-0040420-zotfbuwu
authors: Ridgway, Geoffrey; Taylor, Paul
title: Microbiology and virology
date: 2016-10-14
journal: Basic Science in Obstetrics and Gynaecology
DOI: 10.1016/b978-0-443-10281-3.00011-7
sha: 4e6dae99d5c41d77982f8cf2f06b68b54f38ad09
doc_id: 40420
cord_uid: zotfbuwu

nan

Bacteria are the smallest organisms capable of a freeliving existence. That is, with the exception of a few highly evolved examples, they are able to take up nutrients from the environment, grow and self-replicate independently of other living cells. Their biochemical pathways are similar to those of other organisms, but they are morphologically less complex than the cells of higher organisms. The adjective 'prokaryotic' distinguishes the absence of membrane-bound organelles characteristic of bacteria from the 'eukaryotic' cell characterized by the presence of a nuclear membrane.

The cell wall of acid-fast bacteria such as the mycobacteria and Nocardia spp. contains a high lipid content. They are difficult to stain by most stains, but a solution of hot phenolic carbol fuchsin, or the fluorochrome auramine, which binds to the lipid, will resist decoloration with sulphuric acid, and stain the organism.

The nucleus is a tightly coiled circular double strand of DNA, which replicates by simple fission. Other units of straight or circular DNA termed 'plasmids' may occur loosely in the cytoplasm. These may code for non-essential features such as antibiotic resistance or ability to ferment certain sugars such as lactose. The ability of bacteria to transfer plasmid DNA between bacteria of the same or different species may result in the spread of antimicrobial resistance (plasmid mediated). Bacteria may also transfer genetic material from the nucleus (the so-called 'jumping gene'), leading to stable, chromosomally mediated resistance.

Projecting through the cell wall may be flagellae, fimbriae or pili. Flagellae are long whip-like structures associated with motility. Fimbriae form a fringe around bacteria allowing gliding movement. Pili are longer than fimbriae, and more numerous than flagellae. They are associated with conjugation between bacteria of the same or different species, during which the exchange of genetic material, and hence transferable antibiotic resistance, can occur.

The majority of bacteria are either rod-shaped (bacilli) or spherical (cocci). Cocci may be in chains, e.g. streptococci, or in clusters, e.g. staphylococci. Comma-shaped bacteria called 'vibrios' and the spirochaetes are examples of spirally coiled bacteria. The actinomycetes are the only genus-forming branching filaments. However, in smears, lactobacilli which are morphologically similar may appear to branch, leading to confusion in the evaluation of cervical specimens for actinomycosis.

A few bacteria will produce endospores, a highly resistant resting phase. This is a particular feature of the genera Bacillus spp. and Clostridium spp.

The classification of bacteria is complicated by the lack of clearcut evolutionary relationships between different members. Although the familiar hierarchy of species, genus, family, order, etc. is preserved, it often represents a grouping of organisms with shared characteristics rather than evolutionary relatedness. Knowledge of a simple classification is however important for a number of reasons. It enables communication between scientists, gives a broad picture of how the organism may behave in vitro and in vivo, and may give some indication of the likely efficacy of proposed antimicrobial chemotherapy. Properties used in the classification ing power of an electron microscope is required. Figure  7 .1 is a diagrammatic representation of the internal structures of the prokaryotic cell.

Many bacteria have a capsule or loose slime around the cell wall. This is an important protective mechanism. The ability of organisms such as Staphylococcus epidermidis to produce slime (glycocalyx) on the surfaces of cannulae results in the protection of the organism from the action of antimicrobial agents, and difficulty in eradicating the organism in catheterassociated sepsis.

The cell wall of bacteria is unique. It consists of a backbone of N-acetyl-glucosamine and N-acetylmuramic acid residues linked to polypeptides, polysaccharides and lipids, called 'peptidoglycan'. Peptidoglycan is responsible for the rigidity of the cell wall, and maintenance of the characteristic shape of an organism. Gram's stain differentiates bacteria into those that take up and retain a complex of crystal violet and iodine, and those that do not. This ability is a function of the cell wall. Gram-positive organisms (stained blue/black) have a cell wall consisting largely of peptidoglycan linked to teichoic acids. In contrast, the cell wall of Gram-negative organisms (usually counterstained pink) is far more complex with an outer membrane of lipoprotein and lipopolysaccharide (also unique to bacteria), separated from the peptidoglycan layer by the periplasmic space. This arrangement has important consequences for the ability of Gram-negative bacteria to neutralize the activity of certain antimicrobial agents such as the cell wall active β-lactams (penicillins and cephalosporins). Peptidoglycan is synthesized with the assistance of transpeptidases, also known as penicillin-binding proteins (PBPs), which are a target for β-lactams. This group of antibacterial agents is thus acting against a metabolic pathway unique to bacteria, with consequent low toxicity to eukaryotic cells. The presence of β-lactamases in the periplasmic space may result in the bacteria being resistant to these agents. Mycoplasmas are unique among bacteria in not having a rigid cell wall, while the chlamydiae lack peptidoglycan. Not surprisingly, these bacteria are essentially resistant to β-lactams. of bacteria include: morphology, staining reaction, need for oxygen, utilization or production of various chemicals, chemical constitution and, increasingly, genomic make-up. The latter includes genome size, guanosine and cytosine ratio (GC ratio) and DNA relatedness as determined by hybridization and sequencing techniques.

The naming of bacteria follows the conventional Latin binomial system which is overseen by an international body that applies strict rules. The genus is always written with a capitalized first letter, and followed by the specific epithet commencing with a lower-case letter. Both components are written in italics -thus, Staphylococcus spp. and Staphylococcus aureus. The generic name may be abbreviated after first use, thus S. aureus, or if confusion is likely to Staph. aureus. All other references to specific bacteria are not italicized, including family names such as 'Enterobacteriaceae', trivial names such as 'coliform', or adjectives such as 'staphylococcal'. Table 7 .1 is a simple classification of medically important bacteria based on these characteristics.

In addition to a need to classify bacteria, it is often necessary to distinguish between infecting organisms of the same species, for example when trying to trace the source of a staphylococcal outbreak, or confirming the chain of infection in a case of alleged sexual abuse. A variety of methods are available; some more applicable to some species than others. It is always much easier for bacteriologists to prove that two organisms are different, than the converse.

The protein and polysaccharide components of the bacterial cell are highly antigenic. Differences in the structure of lipopolysaccharides in the cell wall of Enterobacteriaceae (see Table 7 .2) are the basis for somatic or O typing of strains. Capsular polysaccharide antigens are used for K typing and flagella antigens provide the H antigens. Typing using antibodies to H and O antigens is of particular importance in 'speciating' Salmonella spp. The Vi antigen is a further virulence marker particularly associated with S. typhi. Shigella spp. and enteropathogenic Escherichia coli isolates are also typed using antibodies to the O antigens.

Staphylococci are infected with highly host-specific viruses called 'phages'. These phages may transfer genetic material between different staphylococci in a way analogous to plasmid transfer in other bacteria. The pattern of phages infecting a staphylococcus can also be used to demonstrate that the same strain of staphylococcus is responsible for an outbreak. Other methods include biotyping on biochemical features, serotyping based on specific antibodies, antibiograms based on antimicrobial resistance, protein composition (e.g. gel electrophoresis and isoelectric focusing) and plasmid typing. Application of molecular technology has produced highly specific techniques based on 

The distinction between commensal and pathogenic organisms is far from clearcut. Indeed, many of the organisms associated with common infections are part of the normal or transient flora of the body. Mere isolation of the organism from a specimen does not necessarily equate with disease, rather it is isolation of the organism in a site normally sterile. The presence of E. coli in the bowel reflects its normal habitat, but its presence in bladder urine indicates urinary tract infection. Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are all on the one hand normal inhabitants of the upper respiratory tract, and on the other capable of causing lower respiratory tract infection.

Some organisms are always pathogenic to humans. Examples include the plague bacillus, Brucella spp. and Treponema pallidum. At the other extreme are organisms that are usually quite innocuous unless the host's defences are markedly impaired. These are the 'opportunistic' organisms, such as Pseudomonas aeruginosa, often associated with sepsis in the immunosuppressed. These organisms are much more at home in the environment than growing on or in the patient. It is not possible to maintain a biological surface as sterile. The surface (e.g. an initially sterile burn) will soon become colonized with whatever organisms are around. If in addition the biological surface has become selective because of antibiotic administration, the colonizing organism is likely to be resistant to that antibiotic. The concept of creating the selective medium is important; it is, after all, what the laboratory does to select a single organism from a mixture -merely an in-vitro version of what the clinician may unwittingly be doing in vivo.

While a breakdown in the host immune system may lead to commensal organisms causing disease, bacteria have evolved a number of mechanisms to enhance their disease-causing potential, and allow them to evade the immune system. Resistance to lysis by serum is a feature of the Enterobacteriaceae, associated with the presence of lipopolysaccharide at the cell surface. Initial contact with the host may be facilitated by a variety of adhesions. Once attached, the next obstacle will be the host's immune system. The presence of a capsule, with or without antigenic similarity to the host, or the production of a protective biofilm may protect the organism. More sophisticated evasive mechanisms include the production of proteases that cleave IgA, a feature of pathogens invading via mucosal surfaces such as Neisseria spp., or coating with host proteins, such as fibronectin as found in T. pallidum. Chlamydia trachomatis is able to prevent the fusion of lysosomes to the intracellular phagosome containing the infectious elementary body; thus the host protects the invading organism from destruction.

In order to initiate an infection of a clean wound with Staph. aureus, some 10 5 organisms are required. However, the presence of a foreign body, be it traumatic or a medically inserted cannula, reduces the required inoculum by 99% to 10 3 . Such numbers are small by microbiological standards.

Iron is an important growth factor for some bacteria, and they are able to fix iron-binding proteins either by having specific receptors for lactoferrin or transferrin (e.g. Staph. aureus), or by producing extracellular chelators (e.g. some coliforms). Other extracellular products such as hyaluronidase and the ureases of Proteus spp. and Helicobacter pylori may contribute to pathogenesis.

Toxin production is important for the ability of many pathogens to cause disease (virulence). These toxins may be found extracellularly as exotoxins, or released on cell death as endotoxins. Exotoxins are a feature of Gram-positive and Gram-negative organisms. Examples of the action of exotoxins include the neuromuscular effects of Clostridium botulinum and Cl. tetani toxins, gastrointestinal symptoms of cholera, E. coli, Shigella spp. and Staph. aureus, and skin necrosis from Staph. aureus. Some toxins require the infection of the bacteria with a phage for expression, for example diphtheria toxin which affects the heart and lungs, and the erythrogenic toxin of Str. pyogenes (Group A streptococcus). Staphylococcal toxic shock syndrome toxin is a potent pyrogen. Some exotoxins can be formalin fixed to produce toxoids, which are used as vaccines, e.g. tetanus toxoid.

Endotoxin is a feature of the Gram-negative cell wall. It is otherwise known as lipopolysaccharide (LPS). The important component is lipid A, which links the LPS to the outer membrane. Lipid A seems to be responsible for the inflammatory responses associated with the endotoxic shock found in severe Gramnegative septicaemia.

The quality of the specimen is particularly important in microbiology. There is little point in taking a poor specimen and transporting it to the laboratory under less than ideal conditions. At best, the result will be unhelpful, and, at worse, highly misleading. In general, specimens from sites thought to be infected will be collected for microscopy, culture and antigen or genome detection. In addition, serum samples may be sent for antibody determination. While the pressures on a clinician are appreciated, it is important that full clinical details including any current or intended antimicrobial therapy are given. The laboratory will be putting up tests, and interpreting the results in the light of the clinical information supplied.

Specimens should almost always be taken before treatment is commenced. Sensitive bacteria will not survive in the presence of antibiotics, and even if clinically resistant may not be recoverable on artificial media. The correct transport medium should always be used for swabs, to maintain the balance of organisms as similar to the clinical situation as possible, and to ensure the likely survival of pathogens. Because organisms will continue to divide at ambient temperature, specimens should be kept at +4°C and transported to the laboratory as soon as feasible. Some organisms, for example chlamydiae and viruses, survive better at −70°C. The use of a conventional deep freeze at −20°C is satisfactory for preserving bacteria and storing serum samples, but is lethal to chlamydiae. Exceptions to these rules are fastidious organisms such as the gonococcus, which do not survive well out of the clinical situation and should be either direct plated at the bedside or rapidly transported to the laboratory. To increase the likelihood of a positive result, liquid pus should always be preferred to a swab dipped in the pus; gas liquid chromatography which in skilled hands can rapidly discriminate different anaerobes is now rarely used in practice. Different antigen or genome tests require different collection media, even where the same organism is being detected. It is therefore necessary to check with the laboratory before sending these specimens. If the possibility of sexual abuse arises, it is vital to set up a formal chain of evidence with the laboratory, or the evidence may not be admissible in court.

The majority of bacteria are still identified by culture on solid agar media. This means that a minimum of 18 h will elapse before even presumptive results are available. Microscopy will assist in some cases, but where there is a heavy normal flora, such as in the respiratory tract, identification of potential pathogens may be impossible. It is never possible to speciate organisms on microscopy. Thus intracellular Gramnegative cocci are not necessarily synonymous with N. gonorrhoeae, and should never be reported as such until confirmatory results are available. Culture of organisms is necessary in most circumstances to define a full picture of the organisms colonizing or infecting a particular site. Sites that are normally sterile, such as blood and cerebrospinal fluid, should present little problem to the laboratory as any organism ought to be significant. However, the possibility of contamination of the specimen during collection, even under optimal conditions, may make interpretation difficult. The problem is much greater with specimens from a site with normal flora, because, as previously stated, many potentially pathogenic organisms may also be part of the normal flora. Further, it is not yet routinely possible to predict sensitivity to antibiotics without exposure of actively dividing organisms to them.

No microbiological test is 100% sensitive, but the specificity of culture approaches 100%. The same may not be true of antigen-detection systems, although even here the tendency is to concentrate on good specificity over sensitivity. This is because a false-positive diagnosis is more likely to mislead than a false-negative one. In the latter situation clinical impression will override the negative report from the laboratory. Non-culture detection tests provide two useful functions. First, they may be used in situations where rapid diagnosis has important therapeutic and public health consequences, e.g. in meningitis. Second, the tests are useful to diagnose pathogens that are difficult or slow to isolate in the laboratory. The best example of this group is in the diagnosis of chlamydial infection. Because of the need for cell culture to isolate the organism, the development of non-culture detection tests has served to highlight the prevalence and importance of the organism, and also to make diagnostic facilities more widely available. The disadvantage is that the tests are of variable sensitivity, and in some hands specificity is less than optimal. Direct immunofluorescence tests are of good sensitivity, but are subjective; in contrast enzymeimmunoassay systems are of high specificity, but generally of lower sensitivity. The importance of this discussion is that, in low prevalence populations, a low sensitivity (around 90%) may lead to a positive predictive value of under 50%. That is, one in two positive results may be a false positive.

Molecular technology is revolutionizing diagnostic microbiology. Tests based on the polymerase chain reaction (PCR) and the closely related ligase chain reaction (LCR) are now established in the routine diagnosis of certain pathogens such as Neisseria meningitidis and C. trachomatis. These techniques are extremely powerful, and as such are subject to contamination problems. Only validated tests should ever be used for routine diagnostic purposes. Biological inhibitors may reduce the sensitivity of these tests in practice.

Antibody detection tests have the theoretical advantage that all that is required is a sample of clotted blood. Unfortunately, in practice, it is unusual for a definitive diagnosis to be made on a single sample of serum. The antibody rise takes a minimum of 10-14 days, and in some infections, e.g. chlamydial infections, more than 3 weeks may elapse. The safest criterion for the diagnosis of infection using serology is a greater than four-fold rise in specific antibody titre in at least a pair of sera. The exceptions are diseases where antibodies to the organism in question are rare in the normal population, or the organism cannot be cultured. An example of the former is plague, and of the latter syphilis. In the case of syphilis, several different tests are carried out on a single specimen in an attempt to confirm the treponemal infection, and also to define the stage of the disease.

The relationship between humans and their microbes is complex. Products synthesized by one organism may assist the growth of another organism, which may in turn produce factors which will protect the host from invasion by extraneous organisms. Constant stimulation of the host immune system by resident bacteria will lead to early recognition and elimination of related but potentially pathogenic organisms, as well as contributing to the control of potentially neoplastic host cells by virtue of antigens similar to aberrant host ones. Bowel organisms are capable of synthesizing vitamins. The interactions of the various species of organism found on the skin are important for maintaining a healthy integument by production of fatty acids and other substances that inhibit the growth of potential pathogens. Disruption of this delicate balance will result in symptoms; for example antibiotics that affect the normal gut flora will result in a change in the proportion of different bacterial species, with overgrowth of some at the expense of others. This imbalance is manifest by diarrhoea. A more sinister consequence may be the proliferation of Cl. difficile, an anaerobic rod usually present in small amounts, leading to toxinmediated pseudomembranous colitis.

The interaction of aerobic organisms with anaerobic organisms is particularly intriguing. The aerobes serve to consume oxygen, thus lowering the oxygen tension (eH) to very low levels, and allowing the proliferation of strictly anaerobic organisms. The anaerobes outnumber the aerobes by 10 : 1 to 100 : 1 on the skin, rising to over 1000-fold excess in the large intestine. One gram of faeces contains some 10 8 aerobic organisms and 10 11 anaerobic organisms. Maintenance of the anaerobic gut flora is essential for health, and the use of anaerobesparing antibiotics (e.g. ciprofloxacin) where indicated is less likely to lead to diarrhoea as a side-effect.

The predominantly Gram-positive resident flora of the skin is supplemented by transient organisms, usually from the environment, and often Gram-negative. They are unable to establish themselves, but may survive for several hours. This is long enough for transfer to occur to susceptible individuals via the examining fingers.

The normal flora of the vagina changes under the influence of circulating oestrogens. The presence of oestrogen leads to an environment rich in glycogen, which favours the growth of lactobacilli and other acidtolerant organisms. The metabolism of glycogen to lactic acid results in a pH < 4.5. Other bacteria commonly present include anaerobic cocci, diphtheroids, coagulase-negative staphylococci and α-haemolytic streptococci. In addition, a number of organisms that are also potential pathogens may colonize. These include β-haemolytic streptococci including Str. agalactiae, and Actinomyces spp. The balance between health and disease in the vagina is delicate. Factors leading to alteration of this balance will lead to overgrowth of organisms at the expense of the lactobacilli leading to bacterial vaginosis. Specific disease is caused by yeast-like fungi (e.g. Candida spp.), or infection with the protozoon Trichomonas vaginalis. Gonococcal and chlamydial infections affect the cervix, causing upper genital discharge. Bacterial vaginosis, gonococcal and chlamydial infections all predispose to ascending infection resulting in endometritis and salpingitis, with the attendant sequelae of ectopic pregnancy or infertility. Bacterial vaginosis also appears to be a factor in the pathogenesis of pre-term labour.

Gram-positive and Gram-negative bacteria Table 7 .2 lists some of the more medically important bacteria. Staph. aureus is distinguished from other staphylococci by production of coagulase. Increasingly, these organisms are proving to be resistant to the antistaphylococcal β-lactam antibiotics (penicillins and cephalosporins). Such strains are designated methicillin-resistant Staph. aureus (MRSA) after the now obsolete antibiotic used as a laboratory test to detect them. Strains are frequently also multi-resistant, and some are able to spread easily through clinical areas (epidemic MRSA -EMRSA). MRSA are usually no more virulent than other coagulase-positive staphylococci, and frequently colonize wounds and carrier sites. However, when they do cause infection the antibiotic choice is considerably limited compared with methicillin-sensitive strains. Streptococci are divided into three broad groups based on their haemolysis of horse blood agar. Strains producing partial haemolysis (resulting in a greenish pigmentation of the agar) are termed α-haemolytic. This group comprises a number of commensal strains found particularly on the skin and in the mouth ('viridans' streptococci), but they are also important pathogens in deep-seated abscesses and endocarditis. The pneumococcus and enterococci (Enterococcus (Streptococcus) faecalis and Ent. faecium) are also important members of this group. Pneumococci are showing increasing resistance to penicillin. The enterococci are frequent super-infecting organisms, particularly associated with cephalosporin therapy. Glycopeptides (vancomycin and teicoplanin) are often required to treat enterococcal infection; consequently the emergence of vancomycin-and teicoplanin-resistant strains (VRE) is a major worry. Complete haemolysis is termed β-haemolysis. Organisms in this group are further subdivided into the Lancefield Groups A to O. Some α-haemolytic strains also have Lancefield antigens, e.g. the enterococcus is Lancefield Group D. The major human pathogens are in Groups A, B, C and G. However, members of these four groups may also occur as normal human flora. The Group A streptococcus is the most important pathogen (Str. pyogenes) and remains fully sensitive to penicillin. The third broad group is the non-haemolytic streptococci, which are commensal organisms, although anaerobic streptococci may cause wound infections.

The corynebacteria are Gram-positive rods widely distributed over the skin and upper respiratory tract. It is important to differentiate rapidly the pathogenic C. diphtheriae strains from the commensals, and to determine whether the former are toxin-producing strains. C. jeikeium strains have achieved some notoriety by their ability to colonize intravenous cannulae, particularly in the immunosuppressed. Strains are frequently multiply resistant, and may require glycopeptide therapy, or removal of the cannula.

Listeria monocytogenes is of particular importance in obstetrics. It is a motile Gram-positive rod widely distributed in nature. The organism is capable of active division at low temperatures, e.g. in display refrigerators. Depending on regional, occupational and animal exposure, between 5% and 70% of the population carry the organism in the bowel, and strains can be isolated from soil, vegetables, salads and dairy products, and uncooked or partly cooked chicken. Of the 13 serovars, only two are of importance in human disease. Infection in adults is an important cause of meningitis. Maternal commonest bacterial cause of meningitis. Both organisms are capable of causing genital infection. N. gonorrhoeae infects columnar cells; it is therefore a parasite of the cervix, not the vagina. Moraxella catarrhalis strains are usually resistant to penicillins, which may compromise treatment of exacerbations of chronic bronchitis.

The enteric Gram-negative rods comprise a large group of morphologically identical organisms. All are to be found in the gut. The simplest classification divides them into those that ferment lactose, and those that do not. The lactose fermenters include Escherichia coli, Enterobacter spp. and Klebsiella pneumoniae. The nonlactose fermenters include the enteric pathogens such as Shigella spp. and the salmonellae. There are over 2000 types of salmonella, including enteric fever-causing typhoid and paratyphoid, and the common species associated with food poisoning such as Sal. typhimurium and Sal. enteritidis. Other important Gramnegative aerobic bacilli include Pseudomonas spp. and Acinetobacter spp. These are predominantly environmental organisms that will colonize and infect wounds opportunistically -that is, wounds in patients who are debilitated, immunosuppressed or on long-term inappropriate broad-spectrum antibiotics.

The anaerobic Gram-negative bacilli are nonsporing. Although their growth requirements are very precise, they are widely distributed in the body, colonizing bowel, oropharynx and vagina. They may contribute to the formation of abscesses in association either with other anaerobes, or with aerobic organisms.

The precise cause of bacterial vaginosis is unknown. However, the effect is a change in the balance of the bacterial species making up the normal flora. The normally predominant Gram-positive lactobacilli are replaced by Gram-variable coccobacilli. These organisms characteristically adhere to the squamous cells and are called 'clue cells' when seen in vaginal smears. The organisms include the anaerobic Mobiluncus spp. and the microaerophilic Gardnerella vaginalis. The term 'vaginosis' implies that there is no inflammation of the vaginal wall, but a fishy smelling, watery vaginal discharge is produced with a pH > 5.0.

T. pallidum, the spirochaete that causes syphilis, cannot be cultivated in the laboratory. It is also serologically indistinguishable from the spirochaetes that cause yaws and pinta. In consequence, the laboratory can only provide evidence of current or past treponemal infection. It cannot diagnose syphilis. This unsatisfactory state means that, if there is any doubt as to the cause of serum treponemal antibodies, the patient must be assumed to have active syphilis and be treated accordingly. Syphilis in pregnancy will affect the fetus, result-infection usually occurs late in pregnancy, and symptoms range from mild 'flu-like' to chills, fever and back pain and bacteraemia. Neonates infected during pregnancy are ill at or soon after birth. Symptoms are nonspecific, but respiratory distress is common, with bradycardia, jaundice and hepatosplenomegaly; neurological symptoms and skin rashes are also found. The characteristic lesions found in the placenta, and at postmortem examination of infected neonates are miliary granulomata with focal necrosis. Routine macroscopic inspection of the placenta to exclude these macroscopic lesions should be encouraged. Intrapartum neonatal infection will lead to predominantly meningitic symptoms with an incubation period of 5-7 days.

The only bacteria to show branching are the actinomycetes. These are regarded as higher bacteria, with some characteristics similar to those of fungi. The organism occurs in the mouth, gut and female genital tract. The organism may also colonize intrauterine devices. Pelvic actinomycosis is a rare chronic granulomatous disease. The diagnosis can be made by observing the yellow mycelial masses (sulphur granules) in tissue. Symptoms may mimic pelvic neoplasia, and the distinction is important because actinomycosis may be treated with extended courses of appropriate antibiotics such as amoxicillin or co-trimoxazole. Cytologists frequently report Actinomyces-like organisms seen on cervical smears. This statement is not synonymous with actinomycosis. The organisms seen are usually commensal lactobacilli, which are also long Gram-positive rods and may appear to show branching in smears.

Clostridium perfringens is a component of normal bowel flora. Resistant spores are produced under certain conditions, which may survive inadequate disinfection or sterilization. The organism will proliferate in necrotic or poorly perfused tissue, giving rise to gas gangrene. The source is almost always the patient's own flora. Cl. difficile is also found in the normal bowel, in small numbers. Antibiotics may lead to overgrowth of this organism, and production of an exotoxin which gives rise to pseudomembranous colitis. Practically all antimicrobials may lead to this condition, but it is particularly associated with clindamycin, cephalosporins and more recently ciprofloxacin. Neonatal tetanus may be encountered in areas of poor hygiene, acquired via the umbilical stump wound. Cl. botulinum produces a powerful neurotoxin. The disease in adults results from ingestion of the pre-formed toxin, but neonatal botulism may develop from bacteria growing in the gut.

The Gram-negative cocci of medical importance are contained within the genus Neisseria. Both N. gonorrhoeae and N. meningitidis are fastidious organisms, and care is necessary with specimen collection to ensure that the organisms remain viable. The organisms are usually found within inflammatory exudate cells. N. meningitidis is a common nasopharyngeal commensal, and the specific antibacterial activity and low host toxicity (see also Ch. 12). The β-lactam antibiotics comprise two main groups -the penicillins and cephalosporins -each of which contains a large number of members giving an antibacterial spectrum, at least in theory, spanning the bacterial genera of medical importance. Other members of the class include the monobactams and carbapenems (e.g. imipenem). All act selectively on the penicillinbinding proteins unique to the region of the bacterial cell wall. Glycopeptides such as vancomycin and teicoplanin are also important inhibitors of the cell wall construction, preventing incorporation of new units.

The cell membrane structure of all living organisms is very similar, so polymyxins, which are active at the bacterial cell membrane, are toxic to humans and rarely used systemically. The antifungal agents, nystatin and amphotericin B, act on the unique sterol-containing membrane of fungi, but are in themselves also toxic to animals. The azole antifungals block sterol synthesis and are less toxic.

Similarities of the basic metabolic and nucleic acid synthesizing pathways of plants, animals, fungi and bacteria also causes problems of selective toxicity. Consequently, it is necessary to exploit differing enzyme affinities or alternative pathways to kill infecting organisms selectively with minimal adverse effects on the host. The 70S ribosomes of bacteria are different to the 80S ribosomes of mammals, so that antibiotics affecting bacterial protein synthesis are likely to be ineffective against the host's mechanism. Examples include the macrolides (e.g. erythromycin) and lincosamides (e.g. clindamycin), tetracyclines, aminoglycosides (e.g. gentamicin), fusidic acid and chloramphenicol.

Antibiotics can also affect nucleic acid synthesis. Differing enzyme affinities ensure that toxicity to humans is minimized. The quinolones inhibit the α-subunit of bacterial DNA gyrase, preventing supercoiling of the DNA. The ansamycins (e.g. rifampicin) inhibit bacterial DNA-dependent RNA polymerase. Bacteria need to synthesize folic acid in the same way as other organisms. Sulphonamides and trimethoprim act at different points along the folic acid pathway. Bacteria must synthesize folic acid, while mammalian cells require pre-formed folate, and hence are not affected by sulphonamides, which inhibit folic acid formation. Further along the pathway, the reduction of dihydrofolate to tetrahydrofolate requires the action of dihydrofolate reductase. Trimethoprim, the antiprotozoal pyrimethamine and the anti-cancer drug methotrexate all act at this site. Selective toxicity reflects selective affinity for the relevant enzyme.

The actual site of action of nitroimidazole drugs such as metronidazole is unknown. However, the active compound is known to be a reduced form of the drug which is produced only at the very low oxygen tension (eH) produced in the cells of anaerobic bacteria. The ing in a number of characteristic clinical features such as rashes, snuffles, teeth abnormalities, hepatosplenomegaly, proceeding over months and years to osteochondritis and gummata. Specific treatment at any time in pregnancy will result in a healthy neonate.

Mycoplasmas are widely distributed throughout plants and animals. There are more than a dozen species colonizing humans, in the oropharynx, bowel and genital tract. The majority of these strains are commensal, and their role in disease is controversial. Mycoplasma pneumoniae is an important cause of atypical pneumonia. Mycoplasma hominis is found in some 20% of sexually active women, and may be associated with bacterial vaginosis and PID; it causes some cases of pyelonephritis. Ureaplasma urealyticum is present in up to 80% of sexually active women. Its role in disease is less clear. Both U. urealyticum and M. hominis have been isolated from chorioamnionitis. Mycoplasma should be considered as a cause of postpartum pyrexia and treatment with tetracyclines considered if the fever does not settle. M. hominis differs from other mycoplasmas infecting humans by being resistant to macrolides (e.g. erythromycin) but sensitive to clindamycin. Mycoplasma genitalium is difficult to isolate in the laboratory for routine purposes, but there is evidence from molecular studies that it plays a role in pelvic inflammatory disease.

The chlamydiae are among the most sophisticated bacteria known. They are obligate intracellular parasites with a unique lifecycle involving an extracellular transport phase -the elementary body (EB) -and an intracellular phase -the reticulate body (RB). The lifecycle is about 48 h, during which the EB is taken up into a phagosome within the host cell, and transforms into a RB. Division of the RB leads to an inclusion full of daughter RBs, which condense to form the much smaller EBs. Release of the EBs by rupture of the host cell allows infection of further cells. The organisms cannot be cultured on artificial media, requiring living cells. This makes their laboratory isolation inconvenient. Culture has for routine purposes been superseded by antigen detection, e.g. direct immunofluorescence or enzyme immunoassay, or by molecular technology using PCR. Serology is of limited use in the diagnosis of acute chlamydial genital infection owing to cross-reaction of C. trachomatis with the commoner respiratory species C. pneumoniae. As with N. gonorrhoeae, C. trachomatis also infects columnar epithelium, and so is found in cervical cells.

The unique structure of the bacterial cell wall has led to the development of chemotherapeutic agents with penetrates surfaces poorly, or moist heat in the form of pure steam. The process of sterilization by heat requires a heating-up period, a sterilizing time at the correct sterilizing temperature, a further safety period at this temperature, to give a total holding time at the sterilizing temperature, and a cooling period. The entire process time is the cycle time, and will depend on the method of sterilization and the type of load, e.g. an open tray of instruments or a wrapped operative pack containing metal and other materials.

Dry heat is of limited use in surgical practice because it requires a holding time of 1 h at 160°C, giving a cycle time of over 2 h. At this temperature, materials other than metal may char. The use of pure steam is considerably more efficient, requiring lower temperatures for shorter holding times. The basic time/temperature used in the UK is 134-137°C held for 3 min. This equates to a cycle time of some 10 min, and should not be confused with the American standard of 137°C with a holding time of 10 min. Two basic forms of steam sterilizer are in use. The downward displacement autoclave relies on the incoming steam to displace air from the load. Any combination of air and steam will result in sterilizing conditions not being achieved. Therefore a downward displacement autoclave using the UK cycle cannot be used to sterilize wrapped loads or loads with narrow lumens, such as liposuction cannulae. To achieve reliable air removal and steam penetration, a vacuum autoclave is required, which draws a high prevacuum before steam is introduced to the autoclave chamber. It is important that the instruments placed in a downward displacement autoclave are packed loosely, not placed within impervious containers. In contrast a high vacuum autoclave is packed tightly to physically remove the bulk of air in the chamber. Recently, benchtop vacuum autoclaves have been developed. These allow small wrapped loads or a few items with lumens to be processed away from sterile service departments. These machines must not be overloaded. The quality of water used to generate the steam is also important. Water for irrigation should be used in benchtop autoclaves and changed at least daily; this prevents the build-up of pyrogens such as endotoxin, which may remain despite the organisms being killed. It is important that autoclaves are properly maintained, with daily, weekly, quarterly and annual checks being performed relevant to the machine and type of cycle and an audit loop of recording these checks.

Disinfection by heat usually involves the use of machines called washer disinfectors. These are in use for disinfection of crockery, as bedpan washers, and for processing instruments before sterilization. The key is obtaining a temperature of at least 80°C for 1 min. The load is usually heat-dried to avoid the use of drying cloths. action of this active form is thought to be against the nucleus.

Bacterial resistance may be mediated by one of four mechanisms:

1 . The antibiotic may not get into cells, e.g. vancomycin and Gram-negative organisms. 2 . It may be rapidly eliminated by efflux mechanisms, e.g. tetracycline resistance. 3 . Enzymes may destroy the antibiotic, such as β-lactamases and aminoglycoside-modifying enzymes. 4 . The target site may be altered or blocked, such as by rifampicin or quinolone resistance. What is apparent is that the ingenuity of the bacterial cell knows no bounds when it comes to the battle for survival. The antibiotic that has no resistance to it has not yet been discovered. Multi-resistant bacteria are becoming more common, and more difficult or even impossible to treat with currently available drugs.

The technological advances in medicine have resulted in a vast array of different materials being used to manufacture devices for insertion into the body for therapeutic purposes. Ever since antisepsis was first demonstrated to reduce postoperative sepsis by Joseph Lister in 1867, it has been axiomatic that devices should be pathogen free. Antisepsis was replaced by asepsis at the turn of the century, but the comment that is ascribed to the surgeon Berkeley Moyhnihan (1865-1936) that 'every operation in surgery is an experiment in bacteriology' remains as true today as in the 1920s.

Sterilization is the removal of all microorganisms including spores, and is defined internationally as a viable organism count of less than 10 −6 . That is, a single viable organism in one of a batch of 1 million surgical packs would mean that sterile conditions had not been achieved. Disinfection is the removal of all actively dividing organisms, and may not necessarily include spores of fungi or bacteria, nor viruses or prions (such as the spongiform encephalopathy agents). It equates to a reduction in bacterial load in excess of 10 5 . The difference between the two concepts is crucial. Sterilization is not easy to obtain reliably and disinfection may be adequate in some circumstances if done properly. Sterilization is always preceded by disinfection, in order to reduce the bioburden. The three components of disinfection are: (1) cleaning, (2) heat and (3) chemicals.

Heat results in coagulation of proteins and loss of viability. Heat can be in the form of dry heat, which embedded in a matrix of protein and the polysaccharides mannan or glucan.

Most fungi that infect humans grow at a wide range of temperatures, although the optimal temperature for the majority is between 25°C and 30°C. The dermatophytes responsible for skin infections, such as ringworm, grow best between 28° and 30°C, while organisms such as C. albicans or Aspergillus fumigatus, which are responsible for systemic infections, grow best at 37°C. Fungi are predominantly aerobic, but many yeasts can produce alcohol by fermentation as an end-product of anaerobic metabolism. Virtually all fungi have the potential to reproduce by production of asexual spores. These may be conidia, produced in large numbers by moulds, such as aspergillus or the dermatophytes, or the chlamydospores produced in small numbers for survival in extreme conditions by fungi such as C. albicans.

The majority of fungi pathogenic to humans were thought to lack a sexual phase in their lifecycle and were therefore classified as 'fungi imperfecti'. A sexual phase has now been demonstrated in the laboratory for many of these pathogenic fungi, allowing them to be more accurately classified; however, it is convenient in the medical context to leave them under a single grouping of 'fungi imperfecti'.

There are four main groups of pathogenic fungi:

1 . Moulds (filamentous fungi) 2 . True yeasts 3 . Yeast-like fungi 4 . Dimorphic fungi. Most pathogenic fungi are easily cultured in the laboratory, using Sabouraud's dextrose agar, with and without supplements. Candida spp. and many other pathogenic fungi will also grow on blood agar.

These grow as long, branching filaments called 'hyphae', which intertwine to form a 'mycelium'. Reproduction is by spores, including sexual spores, which are characteristic and are important in identification. The fungi often appear as powdery colonies on culture owing to the presence of abundant spores. Included in this group are the dermatophytes, responsible for common superficial skin, nail and hair infections, and belonging to the genera Trichophyton, Microsporum and Epidermophyton, and also the moulds causing systemic infections in the immunocompromised, for example Aspergillus fumigatus or Mucor spp.

These are unicellular, round or oval fungi. Reproduction is by budding from the parent cell. Characteristically, cultures show creamy colonies. The major

The inappropriate use of chemicals is a potential source of infection. Chemicals are incapable of reliable sterilization, except under very carefully controlled circumstances, seldom reached in clinical practice. The term 'high-grade disinfection' describes attempts to achieve chemical sterilization of articles that cannot be sterilized by conventional means. Chemicals are markedly affected by a number of factors, including:

• Spectrum of activity • Temperature of use • Presence of organic debris • Contact time and penetrability • Dilution • Stability at in-use dilution • Inactivators (such as plastics and hard water).

Many disinfectants are odourless and have the 'disinfectant' smell added. 'Pine fluid' has practically no disinfectant action. Cetrimide is widely used in the laboratory as a selective medium for growing P. aeruginosa. It is vital that the correct disinfection process is used for the proposed task. Prior cleaning must always occur. For the processing of endoscopes, this should involve a mechanical washer because cleaning is likely to be more efficient than manually, reducing the chances of biofilm build up in the lumens. All disinfectants are toxic to humans and require care in use. Many disinfectants are corrosive, and it is prudent to ensure that the manufacturer has confirmed that the intended process will not damage the instrument and will be effective in decontamination. The machines used to clean scopes must also be fully maintained to avoid their becoming colonized and recontaminating the scopes at the end of the process.

Ethylene oxide gas may be used to sterilize heat-sensitive devices. The process is difficult to control, and requires a prolonged aeration phase after sterilization. More recently, gas plasma has become practical. Thoroughly cleaned and dried instruments are placed in a chamber with hydrogen peroxide. Low-frequency radio waves are used to generate a plasma, which converts the hydrogen peroxide to lethal superoxide and superhydroxyl ions. The process is suitable for heatsensitive items. Radiation is used to sterilize single-use items such as syringes after manufacture. It has little practical role in medical practice.

Fungi are generally larger than bacteria and are commonly multicellular. Fungal cell walls do not contain peptidoglycan but owe their rigidity to fibrils of chitin T. vaginalis infects the vagina. The organism is sexually transmitted, and although men may become colonized they generally clear the organism from the urethra within a few days. The organism is similar in size to a white blood cell (10-20 µm), and readily identified by flagella movement in wet preparations under a ×40 microscope objective. The organism has three free flagella, and a fourth is embedded in an undulating membrane along the anterior two-thirds of the cell. The organism may cause an irritant, purulent vaginal discharge, with a pH > 5.0. The vaginal wall may be erythematous. In the USA, some 5-10% of men with a non-gonococcal urethritis (NGU) are infected with T. vaginalis. Treatment is with metronidazole.

T. gondii is an intracellular protozoon with a worldwide distribution, causing infection in humans and a wide range of animals. The asexual phase of the organism (bradyzoite) is able to develop in the tissues of a wide variety of vertebrate hosts, including humans. The definitive host is the cat, both domestic and wild cats, in which the sexual cycle occurs in the intestine. Human infection rates may be as high as 90% in some populations. Infection is most often acquired by ingesting bradyzoites in undercooked meat. It may also follow ingestion of oocysts containing tachyzoites resulting from the sexual cycle in the intestine of a cat, which are then excreted in its faeces. Cat litter trays and garden soil contaminated with cat faeces are a likely source to be avoided in pregnancy. After ingestion, the tachyzoites are distributed to many organs and tissues via the bloodstream and invade nucleated cells in all parts of the body and fetus. They multiply within the host cells, disrupting them by producing tissue cysts containing large numbers of slowly metabolizing bradyzoites. Focal areas of necrosis occur in many organs, particularly the muscles, brain and eye. Human infection is usually subclinical but may produce a glandular fever-like syndrome or choroidoretinitis. Transplacental infection may occur during an acute infection in the mother, which may not be diagnosed but may result in serious disease in the fetus. Infection early in pathogen in this group is Cryptococcus neoformans, which has a large polysaccharide capsule. Encapsulated yeasts seen in biological fluids are diagnostic of cryptococcal infection.

Like yeasts, these appear as round or oval cells and reproduce by budding. They also form long branching filaments known as 'pseudohyphae'. Candida is the characteristic genus in this group with C. albicans being the major pathogen. Formation of germ tubes in serum broth distinguishes C. albicans from other members of the genus for practical purposes. C. albicans may be normal flora of the gastrointestinal tract, vagina or skin. Vaginal carriage is increased in pregnancy. Vaginal candidosis (thrush) is a common cause of vaginal discharge. Systemic candidal infection is a feature of the immunosuppressed, or severely ill patient on broad-spectrum antibacterial therapy.

These grow as yeast forms in the body and at 37°C on culture media, and in a mycelial form in the environment or on culture media at 22°C. Histoplasma capsulatum is a well-known member of this group. Infection is usually asymptomatic, but may produce calcified lung lesions. Chronic infection may lead to lung cavities, but a rare acute progressive disease involving widespread infection of the reticuloendothelial cells is usually fatal.

Pneumocystis carinii was originally considered to be an uncommon parasite until, as a result of DNA analysis, it was re-classified in 1988 as an unusual fungus which is very difficult to culture. The human form of Pneumocystis was named P. jiroveci in 2002, although the acronym PCP for the respiratory disease caused has been retained.

These are unicellular eucaryotic organisms. They are able to reproduce by simple asexual binary fission, or by a more complex sexual cycle with the formation of cystic forms. Among the parasitic protozoa, both forms may occur in a single host.

The protozoa of medical importance are usefully classified into three groups: the sporozoa (containing the non-flagellate blood and tissue parasites), the amoebae, and the flagellates (containing the trypanosomes that cause sleeping sickness, Giardia lamblia and T. vaginalis). A list of some medically important species is given in Table 7 .3. The two protozoa of importance in obstetrics and gynaecology are T. vaginalis and Toxoplasma gondii. 

Viruses contain either DNA or RNA as their genetic material, usually as single molecules but never both. In contrast, all other microorganisms contain both forms of nucleic acid. Viral nucleic acid may be either singlestranded (ss) or double-stranded (ds) and the nucleic acid may be in the form of a single piece or it may be segmented, as in influenza and rotaviruses. The nucleic acid content of viruses is very small when compared with that of the cell. For example, influenza viruses have about one-hundredth of the nucleic acid of the cells they infect. RNA viruses (riboviruses) represent the only form of 'life' utilizing RNA as genetic material.

Viruses can only replicate in living cells, which may be of plant, bacterial (infecting viruses being termed phage) or animal origin. The result of infection of a cell is two-fold: first, and most usually, the formation of new virus particles and, second, some change in the cell (often but not always resulting in its destruction). Thus, viruses may establish latent infection in the cells they infect (e.g. the herpes group of viruses, papovaviruses and some adenoviruses). Alternatively, some viruses (e.g. papillomaviruses and the Epstein-Barr virus) may induce malignant transformation in the cells they infect.

The host cell provides the source of all the machinery required for viral reproduction; the invading virus introduces specific information relating to its own structure and constitution, as well as that required to divert cellular mechanisms to viral ends and for the construction of enzymes needed to manufacture viral products. This information is contained, in coded form, in the sequence of bases in the viral nucleic acid. Thus, infection with the virus results in the introduction into the living cell of an infective and foreign nucleic acid with specific biological properties. Once the virus particle has been taken into the cell, the virus merges its identity with it and the whole entity becomes a new and different cell which may be considered as 'a virus-cell complex'.

Details of the method by which different viruses replicate can be found in standard textbooks. In simple terms for DNA viruses, viral messenger RNA is transcribed from the parental virus DNA within the host cell, and codes for the formation of virus-specific proteins. For RNA viruses, the viral genome acts as a pregnancy may result in a stillbirth, or the birth of a live baby with disseminated infection. Features include: choroidoretinitis, microcephaly or hydrocephalus, intracranial calcification, hepatosplenomegaly and thrombocytopenia. Maternal infection during the third trimester can also be transmitted to the fetus, but at this stage of development it usually causes no damage. Controversy surrounds the benefits of antenatal screening. Maternal infection may go undetected unless serological screening is carried out, but a single estimation of antibody may give rise to unnecessary anxiety because of infection before pregnancy began, which carries no risk to the fetus. A rise in the mother's toxoplasma antibody titre during pregnancy or the finding that she has IgM antibodies, indicating recent infection, raises the question of whether to treat the infection, given that treatment does not guarantee the infant will be unaffected, or to terminate the pregnancy even though it is not certain that the fetus has been damaged. Spiramycin (a macrolide) is the drug of choice for treatment of the mother and her fetus.

The helminth parasites of humans belong to three zoologically distinct groups: trematodes (flukes), cestodes (tapeworms) and nematodes (roundworms, e.g. hookworm, Ascaris lumbricoides). None of the infections has particular significance during pregnancy other than as a cause of chronic anaemia with intestinal infection.

The layperson (and some doctors) think of viruses as being 'small germs'. Although it is true that most viruses are indeed very small, size is not a distinguishing feature since some of the larger viruses (e.g. pox viruses) are larger than small bacteria. Some idea of the size of viruses may be obtained by comparing the size of an animal cell to a lecture theatre seating about 200 people; in such circumstances, a polio virus would be about the size of a squash ball, rubella virus the size of a tennis ball, and measles virus the size of a football.

Viruses are distinguished from other microorganisms by their nucleic acid content and method of replication. Microorganisms other than viruses are really cells; they contain both forms of nucleic acid but DNA is their repository of genetic information. They have their own machinery for producing energy and can synthesize their own macro-molecular constituents, i.e. nucleic acid, proteins, carbohydrates and lipids. They all multiply by binary fission. Viruses contain no ribosomes, mitochondria or other organelles; they are template for the synthesis of new viral RNA. Singlestranded RNA viruses are classified as positive or negative strand according to the way in which coding information is stored in the viral genome. With positive-strand RNA viruses, the viral genome is of the same polarity as messenger RNA, and may itself act as messenger RNA, being translated into code for virusspecific proteins. With negative-strand viruses, a complementary RNA copy of the viral genome, or part of it, acts as messenger RNA. One further group of RNA viruses known as reversi viruses replicates by reverse transcription of viral genomic RNA to form a DNA intermediate, from which both messenger RNA and progeny viral genomes are transcribed. This group includes retroviruses, such as the human immunodeficiency virus (HIV), and hepadnaviruses, such as hepatitis B virus (HBV).

Even before negative staining techniques by electron microscopy were available to determine the fine structure of viruses, X-ray diffraction studies indicated that viruses displayed distinct symmetry properties. Because of the limited genetic information available and for reasons of economy, Crick and Watson postulated that the nucleic acid of viruses would code for a virus coat (capsid) consisting of identical subunits arranged in a single repetitive form; negative staining techniques have confirmed these findings. There are two main types of symmetry: cubic and helical. Helical symmetry is generally associated with rod-shaped viruses and cubic symmetry with the more spherical ones.

In its simplest form, a virus consists of nucleic acid and a protein coat, and it is this protein coat which contains the regular assembly of protein molecules. Some viruses, e.g. viruses of the herpes group and myxoviruses (e.g. influenza), are surrounded by an envelope, which is derived from the host cell membrane during release of the virus particles. The capsid consists of numerous identical smaller units, designated capsomeres, which are constant in number and identical in shape. Figure 7 .2 illustrates cubic symmetry and Figure 7 .3 helical symmetry. The nucleic acid and capsid (nucleocapsid) of viruses exhibiting helical symmetry bear a resemblance to a spiral staircase. Each step bears a constant relationship to its neighbours around a central axis which could be represented by the well of the staircase. Cubic symmetry is more complex and describes a group of regular units which have symmetry properties in common with a cube. Specifically for viruses, it includes the tetrahedron, octahedron and icosahedron. Most viruses exhibiting cubic symmetry that infect humans have icosahedral symmetry (Fig. 7.4) . The particle is three-dimensional with 20 identical faces with 12 vertices; each face is in the form of an equilateral triangle. Figure 7 .5 illustrates the fine structure of some of the viruses discussed in this chapter. No satisfactory electron micrographs of the hepatitis C virus have been published to date and, although an electron micrograph of Japanese B virus is not included, it is somewhat similar in its fine structure to the rubella virus. 

An understanding of the nature, including the structure, of viruses is of importance in the diagnosis of viral infections. Viruses may be identified by demonstrating the effect they induce in living cells (cell culture), which can be visualized by low-power light microscopy. Different viruses induce different changes (cytopathic effects) in different cell lines and the virus may be identified by neutralizing the virus infectivity in cell culture by specific antisera.

Whole virus may also be visualized by electron microscopy but high virus concentrations are necessary and electron microscopy cannot distinguish viruses which are morphologically identical within a single group, e.g. different members of the herpesvirus group. Nevertheless, electron microscopy may rapidly identify a herpesvirus from a vesicular lesion, which may be all that is necessary for clinical purposes. Another virus belonging to the herpes group (cytomegalovirus) may be visualized in the urine of congenitally infected infants.

Using specific antibodies, most usefully monoclonal antibodies, the presence of viral antigens may be identified directly from clinical samples. Alternatively, nonstructural proteins may also be identified in clinical samples. Such techniques are used for the identification of respiratory syncytial virus in children with respiratory infections, and cytomegalovirus in the blood and urine of patients with suspected cytomegalovirus infection.

More recently, techniques of considerable sensitivity and specificity have been employed to identify viral nucleic acid. Thus, nucleic acid hybridization and gene amplification techniques (particularly polymerase chain reaction) are now frequently used in diagnosis to identify a number of viral infections, including infections by the herpes group of viruses, enteroviruses, hepatitis C, hepatitis B and HIV viruses. These methods can also be used to quantify the amount of virus in specimens. This is useful for monitoring virus infections in patients who are immunosuppressed or receiving antiviral therapy.

Serological techniques can be used to determine evidence of immunity to viruses, usually by detecting the presence of virus-specific IgG responses. Diagnostically, a significant rise in antibody titre (> 4-fold) between acute and convalescent sera is significant for determination of recent infection. However, more frequently, evidence of current, recent or persistent infection may be detected by a virus-specific IgM response directed towards viral capsid proteins. Such responses are useful in the diagnosis of intrauterine and some perinatal infections, e.g. rubella, cytomegalovirus and parvovirus B19 infections.

Rather than provide basic information on different groups of viruses, attention will be focused on the importance of viruses which may induce severe infections in pregnancy, as well as intrauterine, perinatal and gynaecological infections. The classification and properties of these viruses is shown in Table 7 .4. Some of these viruses, such as the influenza virus, cause classical acute infections, characterized by a rapid onset of symptoms and a brief period of viral replication, followed by clearance of the virus and resolution of symptoms. Naturally acquired infection with a particular strain of influenza A or B results in long-term immunity to that strain, but not those influenza strains which have exhibited major antigenic changes (antigenic shift) or even minor degrees of variation (antigenic drift). Others cause persistent infections, in which the patient often remains infected for life. Persistent infections may be characterized by an acute phase of infection, which may or may not be symptomatic, followed by life-long latency, where the virus persists in a nonreplicative form with restricted viral gene expression. Subsequent reactivations of infection may occur, although in the immunocompetent person reactivated infection is usually more limited than primary infection, and may be asymptomatic. This pattern of persistence is typical of herpesviruses such as herpes simplex virus (HSV) and varicella-zoster virus (VZV). Other persistent viral infections such as HIV and hepatitis B and C viruses (HBV and HCV) are characterized by ongoing virus replication and chronic, evolving disease.

The features of these viral infections, together with preventive measures where applicable, are listed in Table 7 .5. Some infections may be prevented by immunization, e.g. influenza A and B, and poliomyelitis, and recombinant-derived vaccines are under trial for the hepatitis E virus, which carries a high mortality rate among pregnant patients in developing countries. Although there is some doubt as to whether varicella is more severe in pregnancy, infection is often severe and occasionally fatal among adults generally, particularly those who smoke. Thus, pregnant women who give no history of varicella, or in whom screening tests for VZV antibodies indicate susceptibility, should be protected by the administration of varicella-zoster immune globulin (VZIG) within 72 h of an exposure. Aciclovir treatment should also be used for pregnant women with established infection as they are at Vaccine available on named-patient basis for travellers to endemic areas increased risk of varicella pneumonia, and this has a high mortality rate. Japanese B encephalitis is one of the more widely distributed arbovirus infections, being present in Asia. Although subclinical infection is common, those exhibiting clinical features may experience a mortality rate of up to 20% in outbreaks. Fetal death is common. An inactivated vaccine is available on a 'named-patient basis', but since it may be reactogenic is not recommended in pregnancy. Lassa fever may be particularly severe in the latter stages of pregnancy, and the fetal death rate is high.

Severe acute respiratory syndrome (SARS) is caused by a coronavirus that first emerged in the southern Chinese province of Guangdong in November 2002. Pregnant women with SARS appear to have a worse prognosis and a higher mortality rate. Therefore early delivery or termination of pregnancy should be considered in those who are seriously ill. The following criteria for early delivery have been proposed by Wong et al (2003 There seems to be no reason for elective pre-term delivery in those women who are relatively well with SARS infection. Pregnant women should be treated empirically since a laboratory diagnosis may be prolonged. It has been suggested that the treatment of pregnant women with SARS should be without the use of ribavirin. Infections due to other coronaviruses are relatively mild and have not been reported as causing problems during pregnancy.

Viruses which may damage the fetus are shown in Table 7 .6. The rubella virus, and two viruses belonging to the herpesvirus group -cytomegalovirus (CMV) and varicella-zoster virus (VZV) -as well as human parvovirus B19 may induce persistent infections in the fetus.

tion. In contrast with rubella, primary maternal CMV infection is often asymptomatic, but may result in fetal infection and damage throughout pregnancy. The viral transmission rate to the fetus is of the order of 30-40%, but fetal damage occurs in only about 10% of infected conceptuses. Nevertheless, the burden induced by congenitally acquired CMV infection is considerable; it has been estimated that somewhere in the order of 300-400 CMV-damaged babies are born in the UK each year. CMV is the commonest microbial cause of psychomotor retardation, although deafness may be the sole manifestation of congenitally acquired disease. Recurrent CMV infection or reactivation is rarely associated with fetal damage.

Although very few indigenous adult women born in the UK are susceptible to varicella, the proportion may be considerably higher -up to 35% -among those born and brought up in rural areas of developing countries. The overall risk of congenitally acquired disease following maternal varicella is restricted to the first 20 weeks of gestation, but, in contrast to rubella and CMV, the risks are low (about 1% overall); the incidence is greater between 13 and 20 weeks of gestation (2%) than between 1 and 12 weeks (0.4%). Defects involve the CNS and musculoskeletal system; limb hypoplasia and cicatricial scarring may be present.

If acquired towards term, the infant may develop varicella after delivery. If maternal varicella occurs 8 days or more before delivery, neonatal varicella is usually mild. In contrast, maternal varicella infection

As a result of immunization programmes against rubella, now being directed against pre-school children of both sexes and rubella-susceptible adult women, only about 2% of women of childbearing age born and brought up in Britain are susceptible to infection. However, susceptibility rates equivalent to or higher than those observed in developed countries during the pre-vaccination era are present in many developing countries. Congenitally acquired rubella is now rare in Britain and most industrialized countries, although rubella-induced defects have been reported with varying frequencies in other parts of the world.

Rubella virus produces an anti-mitotic protein and consequently, if infection occurs during the critical phase of organogenesis (i.e. during the first 8 weeks of pregnancy), severe and multiple defects are likely to occur. If infection occurs during the first trimester, fetal infection is almost invariable, and 75-80% of conceptuses are damaged. After the first trimester, the incidence and spectrum of defects is much less. Although congenital heart disease, eye defects (particularly cataracts) and deafness are the commonest manifestations of congenitally acquired infection if maternal infection is acquired in early pregnancy, rubella induces a generalized and persistent infection with multi-organ involvement, and a wide spectrum of defects may be present at birth or evolve in infancy.

About 40-50% of women of childbearing age in Britain have no serological evidence of previous CMV infec- 

Viruses which may cause severe infection if acquired perinatally or during the neonatal period are listed in Table 7 .7. A range of diagnostic methods may need to be employed to confirm viral infection in such cases including qualitative and quantitative molecular techniques.

About 75% of genital infections are caused by HSV-2 and about 25% by HSV-1. Infants may be infected by maternal genital lesions, fetal scalp monitoring, maternal non-genital lesions or contact with HSV-infected nursery staff or visitors. Primary maternal lesions carry a much higher risk of infection than recurrent lesions, that occurs less than 1 week before delivery may be severe and, without treatment, occasionally fatal. VZIG should therefore be given to infants whose mothers develop varicella 8 days or less before delivery; aciclovir may be given if neonatal infection is severe, despite administration of VZIG. Varicella-susceptible pregnant women exposed to infection during the last 3 weeks of pregnancy should be given prophylactic VZIG.

About 40% of women of childbearing age in Britain are susceptible to parvovirus B19 infection. Human parvovirus may induce a rubella-like rash, sometimes accompanied by arthralgia, although infection may also be asymptomatic. The fetus is infected in about 33% of cases, and in about 10% of these spontaneous abortion may occur, usually in the second trimester. Parvovirus B19 binds to a globoside (P antigen) expressed on the membrane of erythrocytes and fetal heart, and this results in a reduction of fetal erythroid progenitor cells, which may result in a severe fetal anaemia, leading to heart failure and development of hydrops fetalis. Heart failure may also result from viral myocarditis. However, developmental defects have not been recorded. Parvovirus infection is therefore not a reason for therapeutic abortion. Fetal anaemia and hydrops may be 'rescued' by fetal blood transfusion.

HIV-1 and -2 WHO estimates that, globally, 38.0 million adults and 2.3 million children were living with HIV at the end of 2005. In developing countries, infection is usually contracted heterosexually. In Britain, HIV infection tends to be concentrated in London. In its inner-city areas, up to 0.5% of pregnant women are now HIV-1 positive. In the absence of treatment with a combination of antiretroviral drugs, HIV-1 is transmitted to the fetus of infected mothers in about 12-15% of cases. Combination antiretroviral therapy has reduced the HIV transmission rate, and studies suggest that chemotherapy together with delivery by caesarean section further reduces the risk of transmission to 1-2%. Infection may be transmitted in utero but occurs more frequently during delivery, or when breastfeeding. In contrast to HIV-1, HIV-2 is transmitted in only about 1% of cases, and this is almost certainly a manifestation of the much lower maternal viral load present. If HIV infection occurs in utero, it is usually possible to establish a diagnosis during the first few weeks of life. If infection occurs during delivery or via breastfeeding, or in infants born to mothers on antiretroviral treatment, it may take considerably longer to establish a diagnosis of HIV infection in infancy. Diagnosis of HIV infection in infancy is usually made by detecting the virus by whether using molecular methods to detect HBV DNA in mothers with anti-HBe may detect those with high levels of viraemia, whose children should be given active/passive vaccination.

It is estimated that there are about 170 million HCV carriers worldwide, relatively high carrier rates (2.5-5%) occurring in some developing countries, particularly in sub-Saharan Africa, Asia and Latin America. In Britain, infection is common among multi-transfused persons, injecting drug users, and those from countries with a high prevalence. The prevalence among pregnant women in some inner-city areas in London is about 0.25%. Infection may be transmitted in utero if acute maternal infection occurs in the last trimester of pregnancy, but mothers who are carriers may also occasionally transmit in utero since HCV RNA has been detected in neonates at birth, and caesarean section may not prevent transmission. Neonatal infection occurs in about 6% of infants delivered of mothers who are HCV carriers and who are HCV RNA positive, but in mothers co-infected with HIV the transmission rate is 30-35%. Mothers who are HCV antibody positive but HCV RNA negative are very unlikely to transmit infection. HCV-infected infants are likely to develop persistent HCV infection which may in due course result in chronic liver damage.

About 100 different genotypes have been identified, of which at least 30 are found in the genital tract. HPV types 6 and 11 cause genital warts, and are known as 'low risk' types as they are rarely found in cancers. HPV types 16, 18, 31 and a few other types are designated as 'high risk' as they are associated with premalignant and malignant cervical disease; viral DNA can be detected in ~95% of cancers, often integrated into host cell chromosomes, and virus-encoded oncoproteins, which bind to and inactivate the p53 and pRB tumour suppresser proteins, are expressed. HPV 6 and 11 may be transmitted from mother to infant at delivery and may cause juvenile laryngeal or genital warts, but this is rare. High-risk types may also be transmitted at birth and may persist in infancy, but they are not associated with obvious disease and the consequence of these infections is unknown. Girls aged 12-13 years are now vaccinated with HPV vaccine to protect against cervical cancer.

This virus is endemic in South-West Japan, the South Pacific, parts of West Africa, the Caribbean basin, southern USA and parts of South America. Persons who have emigrated from these areas may also be car-since primary infections are associated with high concentrations of virus over a long period.

The incidence of neonatal herpes in Britain is estimated to be of the order of 1.6 per 100 000 deliveries, whereas in Sweden and USA it is considerably higher (5 and 7 per 100 000, respectively). The presence of maternal lesions at or within 6 weeks of birth is an indication for caesarean section provided membranes are intact, or ruptured less than 6 h before delivery. Infants delivered via an infected birth canal should be given prophylactic aciclovir intravenously. Although it is recommended that women with evidence of a recurrent lesion at delivery should deliver by caesarean section, transmission is rare; studies from the Netherlands have shown that the risks of acquiring neonatal HSV following caesarean section and vaginal delivery are not significantly different. Testing mothers with a history of recurrent herpes, or whose partners give a history, is no longer recommended, since virus shedding in late pregnancy does not correlate with transmission to the neonate. There is some evidence to suggest that treatment of mothers with oral aciclovir who have a history of recurrent genital herpes during the last month of pregnancy may reduce the incidence of lesions at delivery and consequently the necessity for caesarean section.

Clinical manifestations may be delayed until 10-14 days after birth. Infants may present with lesions of the skin and mucous membranes (60% will disseminate), CNS involvement or generalized infection.

There are 350-400 million HBV carriers worldwide, the highest rates being in South-East Asia (~15%) and sub-Saharan Africa (~10%). In some inner-city areas in Britain, the HBV carrier rate among pregnant women is about 1%. Pregnant women with acute HBV infection are likely to transmit infection to newborn infants perinatally. Infants delivered of mothers who are HBV surface antigen (HBsAg) and 'e' antigen (HBeAg) positive should be protected by the administration of hepatitis B immune globulin (HBIG) and HBV vaccine (active/passive immunization) at birth. Provided a full course of vaccine is given (three doses and a booster), this procedure will effectively reduce the risk of persistent HBV infection in the infant by about 95%, thereby reducing the risk of long-term chronic liver damage and primary hepatocellular carcinoma. Infants delivered of mothers who have antibody to HBeAg (anti-HBe) should be given HBV vaccine without HBIG. Infants whose mothers are HBsAg positive without 'e' markers, or where the 'e' marker status has not been determined, or whose mothers had acute hepatitis B during pregnancy, should be given active/ passive immunization. There is currently a debate on riers. The prevalence of antibodies among antenatal patients in London and Birmingham is 0.14-0.26%. Studies in Japan and the Caribbean have shown that this virus is transmitted via breast milk. Of the carriers of this retrovirus, 2.5-4.0% who have not acquired infection through blood transfusion may develop adult T cell leukaemia or tropical spastic paraparesis 10-30 years after infection.

Severe acute respiratory syndrome and pregnancy

Cytomegalovirus is subclassified as a betaherpesvirus. These herpesviruses usually have a restricted host range and grow slowly in cell culture; infected cells often show cytomegalic inclusions both in vivo and in vitro. They establish latency in a variety of tissues including secretory glands, the kidney and lymphoreticular cells.