Many bacteria and higher eukaryotes live in harmony in a symbiotic relationship that benefits one or both of the partners. Indeed, we are colonised by bacterial cells which outnumber our own cells ten to one. This amicable bacterial lifestyle contrasts with a pathogenic one, in which the bacterium causes damage to its host. This is a potentially dangerous strategy for a bacterium, because the provoked host is capable of fighting back. A pathogenic lifestyle offers short-term gain. By outcompeting other bacteria within its host, the bacterium can achieve local dominance and, by more widespread colonisation, expand its territory more globally. However, evolution has to balance these advantages against the possibility that the bacterium is eliminated. The latter could occur if the bacterium is too weak to prevent its destruction by the strong host defences it has incited or if its potent virulence wipes out the host and, thus, its source of food.

As pathogenicity appears to be such a risky business, it may be an abnormal condition. Evidence to support this view comes from several lines. First, many of the genes involved in virulence appear to be relatively new – that is, new to the organism made pathogenic by their presence. These genes are frequently found on mobile genetic elements such as plasmids, phage, and pathogenicity islands that have recently been acquired by the organism.

Upon infection, pathogenic bacteria must evade the immune defence of their host in order to multiply. To this end, many bacteria secrete toxins as part of their virulence mechanism. In a classical view, toxins are molecules that cause intoxication upon their release by bacteria into the body fluids. However, in the last 10 years a different class of bacterial toxin has been recognised. These molecules are not simply secreted by the bacterium, but instead they are delivered directly from the bacterial cytoplasm into the cytoplasm of the eukaryotic cell by specialised secretion machines present exclusively in Gram-negative bacteria. These are the so-called type III or type IV secretion systems, depending on whether they use a structure resembling the flagella or conjugative pili, respectively. In this chapter, we will describe the mode of action of toxins delivered by type III secretion systems (TTSSs). These molecules, currently known as type III effectors, have been shown to act on different host signalling pathways controlling a number of responses, and in some cases interfere with cell growth.

TYPE III SECRETION SYSTEMS

TTSSs are present not only in bacteria that are pathogenic for animals but also in bacteria pathogenic for plants or even in symbionts for plants and insects (Cornelis and Van Gijsegem, 2000). We will restrict our analysis to the action of type III effectors of animal pathogens. Among these, type III effectors have been identified in Yersinia spp., in Salmonella spp., in Shigella spp., in enteropathogenic and enterohaemorrhagic Escherichia coli, in Pseudomonas aeruginosa, and more recently, in Burkholderia pseudomallei (Stevens et al., 2003).

Although cytomegalovirus (CMV), hepatitis B, C, D viridae (HBV, HCV, HDV), human T-lymphotrophic virus (HTLV) and human immunodeficiency virus (HIV) are not associated with genital tract manifestations, they may be considered as sexually transmitted diseases. These agents pose a particular risk in assisted reproductive technologies because of their potential for transmission to staff, to other patients, and to children born as a result of treatment, particularly via cycles that involve the use of donor gametes. They are responsible for a wide spectrum of clinical illnesses involving multiple organ systems, and all have been recognized to be more prevalent in populations attending STD clinics than in the general population.

The infectious agents of CMV, HIV, HTLV, HBV, HCV and HDV can persist in the blood unknown to the carrier, who may be asymptomatic; they can also be transmitted via other body fluids, including:

• cerebrospinal fluid (CSF)

• pleural fluid

• breast milk

• amniotic fluid

• peritoneal fluid

• pericardial fluid

• synovial fluid

• vaginal secretions

• semen

• any other fluid containing visible blood, as well as all unfixed tissues, organs, and parts of bodies.

Blood-borne virus (BBV) transmission can occur via:

• sexual intercourse

• sharing needles/syringes

• re-use of unsterilized or inadequately sterilized injection equipment

• skin puncture by blood-contaminated sharp objects such as needles, instruments or glass

• childbirth – before or during birth, or through breast feeding

Routine screening

Prevalence of BBV: geographic distribution

The risk of introducing infection into the laboratory can be minimized by screening patients for infectious agents where indicated by medical history and physical examination. The risk of infectious agent transmission in ART procedures varies in different populations and geographical regions; a risk assessment should be carried out according to the prevalence of disease in the specific patient population, bearing in mind the possibility of ‘silent’ infection prior to detectable seroconversion. Figures 15.1–15.4 illustrate the current geographic distribution for infections due to HIV/AIDS, HBV and HCV.

National and international guidelines in many countries now recommend that patients presenting for ART procedures should undergo routine testing for Hepatitis B and C annually; screening for HIV-1 and HIV-2 should be carried out within 3–6 months of starting treatment to allow for the lag time to seroconversion. Human T-cell lymphotrophic virus (HTLV-I and HTLV-II) has a low prevalence in Western countries, with HTLV-I principally endemic in Japan, Central Africa, the Caribbean and Malaysia, and HTLV-II prevalent in Central America and the southern USA. Screening for these viruses prior to blood or organ donation is now mandatory in some countries; guidelines for HTLV-I and HTLV-II patient screening prior to ART procedures should be adapted to local regulations and epidemiology. Routine screening for genital infections, i.e.

Introduction

Although all species of microorganisms discussed so far can be described as ‘parasites’, the term traditionally refers to parasitic protozoa, helminths (worms) and arthropods.

Parasites are divided into two classes.

Unicellular: protozoans (Kingdom Protista); four phyla (see Table 6.1a).

Multicellular: helminths – flatworms (flukes and tapeworms) and roundworms

Arthropods – have chitinous exoskeletons with jointed appendages (lice, mites, ticks, etc.).

Classification of parasites is based upon taxonomic groups and the body site infected. Diagnosis of parasite infections may be made by fecal examination for ova and parasites, antigen detection systems, serologic tests and examination of thick and thin blood films. Molecular techniques for the detection and identification of parasitic infections are also under development.

Terminology

Parasite life cycles and their interrelationship with hosts are described by specific terms:

host: an organism that harbours or nourishes another parasite,

definitive host: harbours the adult or sexual form of the parasite,

intermediate host: harbours larval or asexual stages of the parasite,

reservoir host: a non-human host that can maintain the infection in nature in the absence of human hosts.

parasitism: the parasite benefits at the expense of the host.

endoparasite: lives inside the host, e.g. intestinal parasites, cause infection,

ectoparasite: lives on the surface of the host, e.g. pubic lice, cause infestation.

symbiosis: close association of two species that are dependent on one another.

Pathology in the male upper GU tract can be due to epididymitis, orchitis and prostatitis, i.e. inflammation of the epididymis, testis and prostate. In the female, salpingitis, oophoritis, endometritis and ‘pelvic inflammatory disease’ (PID) describe infection or inflammation of the fallopian tubes, ovaries, endometrium, and pelvis.

Male upper GU infections

Epididymitis

The epididymis, which lies on the posterior surface of the testicle, is responsible for transport, storage, and maturation of sperm. Epididymitis is the most common cause of intrascrotal inflammation, and is likely to be due to retrograde extension of organisms from the vas deferens. Hematogenous spread is very rare. It is primarily a disease of adults, most commonly in the age range of 19–40 years. The onset of pain and swelling is usually gradual, and there may be initial abdominal or flank pain because the inflammation typically begins in the vas deferens. Younger patients may have symptoms of urethritis, and there may be a urethral discharge. A recent history of instrumentation or urinary tract infection is more common in the older patient. The disease is usually unilateral; bilateral involvement is seen in only 10% of cases. Peritubular fibrosis can lead to occlusion of the ductules and obstructive azoospermia. Progression of the infection can lead to an epididymal abscess, epididymo-orchitis, or testicular abscess.

The causative agent can be identified in 80% of cases, varying with the age of the patient.

Genital ulcer disease is linked to an increased risk of HIV transmission in both homo- and heterosexual practice, and therefore the 1990s has seen its emergence as a very important sexually transmitted disease process. Whereas syphilis has been known for centuries to cause significant disease in the sexual contacts and offspring of infected patients, other agents producing soft ulcers such as chancroid and herpes simplex have now increased in prevalence. The incidence of genital ulcer disease caused by syphilis plummeted in developed countries following the availability of penicillin, and the other diseases have increased in significance. Some of these diseases can be managed by antibiotic treatment, but others such as herpes simplex virus (HSV) persist for life as a latent infection with the potential for intermittent reactivation to active disease. Education targeted towards limiting the spread of these agents and the identification of treatment modalities has become an integral part of curbing the HIV epidemic worldwide.

Five different microbial agents are commonly associated with genital ulcer disease:

1. Herpes simplex viruses (HSV-1 and HSV-2)

2. Treponema pallidum: syphilis

3. Haemophilus ducreyi: chancroid

4. Invasive serovars of Chlamydia trachomatis, causing lymphogranuloma venereum (LGV)

5. Calymmatobacterium granulomatis, causing Donovanosis

The prevalence of the different types of disease varies dramatically in different populations.

Rigorous attention to maintaining a clean laboratory environment, a sterile culture microenvironment and aseptic technique at the interface of the two, should minimize exogenous contamination of gamete handling and culture systems in the ART laboratory. All human tissue and samples, regardless of prior testing for infectious agents, must be regarded as a potential hazard and handled accordingly. Samples identified as a biohazard after screening must be handled with extreme caution, according to specified protocols and procedures.

Blood-borne viruses

The blood-borne viruses (BBV), Hepatitis B, C, D and HIV are all potent pathogens capable of causing severe disease and death, with few effective treatments available. Nosocomial transmission of Hepatitis C has been reported (Lesourd et al., 2000, see below), and HBV transmission through contact with environmental surfaces has been demonstrated. The stability of HIV and HBV under different conditions has been determined experimentally.

• HIV in high starting concentrations can remain infectious for 3 weeks or more in dried blood.

• Infectious HIV can be detected for at least 2 months in liquid blood at room temperature.

• HBV in dried plasma can retain infectivity for at least 4 months, and this is likely to be even longer in liquid blood or plasma.

• Infectious HIV has been recovered from bodies up to 16 days after death, and it should be assumed that HBV is at least equally as robust.

The Family Papovaviridae has two genera:

1. papillomaviruses that produce warts in mucocutaneous sites;

2. polyomaviruses, the first class of DNA virus that was recognized to cause tumours in animals, including JC virus, BK virus and SV-40.

Papilloma viruses are non-enveloped DNA viruses with icosahedral symmetry, measuring 45–55 nm in diameter. The human papilloma viruses (HPV) represent a group of over 80 viruses that infect epithelial cells to produce warts in a number of sites: more than 30 types infect the genitalia. Over 90% of all HPV infections are caused by two low-risk (nontumorogenic) strains of HPV, serotypes 6 and 11. These are usually associated with the visible genital lesions and may also be associated with laryngeal and upper respiratory tract condylomata. Other lowrisk HPV serovars include 40, 42, 43, 44, 54, 61, 70, 72 and 81. High-risk serovars include HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82. Overall, HPV16, 18, and to a lesser extent 31, 33, 35, 45, 53 and 58 are the most common high-risk papilloma viruses causing infections in humans. HPV 16 infection represents about 50% of those infections associated with progression to carcinoma.

Genital warts and cervical cancer

Genital human popillomavirus infections (HPV)

Since genital HPV infections are not reportable to the CDC, the real prevalence and numbers of new infections is not really known.

Wolbachia are gram-negative, intracellular bacteria that shelter in the gonads of invertebrates, and have profound effects on the fertility of their hosts. In some species, infected hosts can only reproduce parthenogenetically, in others cytoplasmic incompatibility prevents infected males from breeding with uninfected females, and in some cases genetically determined male embryos are transformed into females. Wolbachia engineers effects, as do all parasites, for selfish ends. Although this bizarre pathology is unknown in medical science, the relationships between microbes and human fertility are nonetheless complex, fascinating and important for the practice of reproductive medicine.

Unfortunately, and usually without advance warning, microbes occasionally enter the clinical laboratory through infected semen or vaginal tissue. When this occurs, a patient's treatment outcome may be seriously compromised because microbes can quickly deplete nutrients in culture media and alter the pH, and it would be irresponsible to knowingly transfer an infected embryo or semen to a patient. Bacterial and fungal growth are often obvious and easily tested, but how often do infectious agents go unrecognized and contribute to the problems of infertility, treatment failure and even possibly affect the child-to-be?

This is the first book on medical microbiology that has been written by experts in reproduction for clinical scientists and physicians in their own field. They are to be congratulated on filling a gap in the literature between microbiology and assisted reproduction, which they achieve in three sections.

The world of microbes is intrinsically fascinating. Microbes are abundant in every place on earth where larger living creatures exist, and they can also thrive in habitat extremes where no other kind of organism can survive for long: from deep under the sea to the stratosphere – up to 32 km in the atmosphere, in oil formations and in hot telluric water. It is estimated that the total biomass of microbes probably exceeds that of all the plants and animals in the biosphere. This biomass is predominantly composed of bacteria, and these microorganisms play a crucial role in recycling much of the organic material in the biosphere. Despite their minute size, microorganisms carry out all the fundamental processes of biochemistry and molecular biology that are essential to the survival of all living species. Although their size may give them the illusion of being ‘primitive’, their range of biochemical and biophysical capabilities is far wider than that of higher organisms. One of their most important properties is adaptability and versatility, a key feature in their long history of evolution. Fossil records suggest that at least some members of the microbial world, oxygen-producing cyanobacter-like organisms, had evolved 3.46 billion years ago (Schopf, 1993); a viable fungus, Absidia corymbifera, was recovered from the right boot that accompanied the frozen, well-preserved prehistoric corpse, ‘Ice Man’, aged approximately 5300 years (Haselwandter & Ebner, 1994).