too use purified PI as vaccinogen. The results of immunisation with PI in animal studies were howeverr disappointing (110), showing the same lymphocyte ...
CHAPTERR 1
Generall Introduction
rr
Introduction Introduction
GENERALL INTRODUCTION 1.. Description of the organism 1.11 History Inn 1944 Eaton and colleagues described an agent that passed through filters with pore sizes thatt retain bacteria and caused pneumonia when inoculated in rodents (1). The agent was believedd to be a virus as it passed through bacteriologie filters and could be grown in chicken embryoss only. The relation of the agent to the primary atypical pneumonia (PAP) syndrome waswas suggested by the fact that human serum from patients recovering from PAP neutralised thee agent. In the early 1960ies it was established that the organism had many characteristics in commonn with the pleuro pneumonia-like organisms (PPLO) isolated in 1898 by Nocard and co-workerss and causing contagious pleuropneumonia in cattle. Its mycoplasmal nature was establishedd by growing the agent on cell free medium (2), and subsequently it was appropriatelyy named Mycoplasma pneumoniae. 1.22 Taxonomy M.M. pneumoniae is one of the currently recognised 102 Mycoplasma species in the order of Mycoplasmatales.. Thirteen of these 102 Mycoplasma species have been isolated from humans.. Mycoplasmas are classified in the Class Mollicutes (mollis=soft, cutis = skin) which namee originates from the lack of a rigid cell wall, separating mycoplasmas from other bacteria.. They are bounded by a cell membrane containing sterols. Because of their deformablee membrane and small size (150-250 nm), mycoplasmas are able to pass through bacteriologiee filters. As mycoplasmas lack a typical bacterial cell wall, they are not visible on Gramm staining and resistant to cell wall-active antimicrobial agents such as penicillins and cephalosporins.. Mycoplasmas are able to grow in cell free media and belong to the smallest self-replicatingg organisms with genome sizes of 580-1350 kb. Recently, sequence data from thee entire genomes of M. pneumoniae (3) and M. genilalium (4) became available. This informationn will enhance the knowledge of the molecular biological basis of these microorganisms.. The small genome is remarkable for such successful pathogens. The question iss whether there is any relation between genome size and the ability of mycoplasmas to grow inn vitro. Recently, Hutchison el al. sorted out the number of essential genes necessary for replicationn of M. genilalium (5). Their conclusion was that 140 of the 340 genes, at least under laboratoryy conditions, are not a prerequisite for the propagation of this smallest-known freelivingg micro-organism. In addition, obligate intracellular bacteria like Chlamydia pneumoniae, CoxiellaCoxiella burnetii, and Rickettsia species have genomes larger than many of the self-replicating mollicutes,, making a direct relation between genome size and the ability to grow in vitro unlikely.. The genome sequence projects provided the genetic explanation for the requirements
99
ChapterChapter 1
off serum, peptone, and yeast extract in nutritious growth media for the in vitro growth of mycoplasmas.. Only a limited number of genes allows biosynthetic pathways in mycoplasmas andd no genes are present for amino acid synthesis (3,4). Therefore, M. pneumoniae and M genitaliumgenitalium are totally dependent on exogenous supply of omino acids. M.M. pneumoniae is a filamentous organism, about 10x200 nm in size, with a genome of 816 kb.. Mycoplasmal DNA has an exceptional low guanine and cytosine (G&C) content, with M. pneumoniaepneumoniae
showing the highest G&C content of 40 mol% of the entire genome.
Characteristicc for mycoplasmas is the use of TGA as a tryptophan codon instead of a stop codon.. The flask shaped cells of M. pneumoniae have a terminal tip structure and exhibit glidingg motility on solid surfaces (6,7). The terminal tip structure is responsible for attachment off the organism to cell membranes. Of the surface proteins required for cytadherence, the 169 kDaa protein PI has been most extensively studied (8-11). 1.33 Habitat Humann mycoplasmas colonise mucous membranes in the ororespiratory or genitourinary tract,, where some species behave as commensals. Under immune suppression, for example afterr organ transplantation, in AIDS patients or in patients with hypogammaglobulinacmia, isolationn of mycoplasmas from joints, pericardial tissue or from respiratory tract tissue differentt from their usual habitat has been described (12,13). Humann mycoplasmas have been shown to be taken up by polymorphonuclear leukocytes (PMNL)) and macrophages (14), but whether mycoplasmas can enter epithelial cells is unclear. M.M. fermentans and M. penetrans have been found in nonphagocytic cells in AIDS patients (15,16).. The mechanism of cell entry is not fully understood. The specialised tip structure of M.M. genitalium and M. penetrans seems to play a role in cell entry but also mycoplasma species lackingg a tip structure like M. fermentans and M. hominis have been found intracellular by electronicc microscopy (17). Whetherr mycoplasmas replicate intracellular^ remains to be resolved. There are indicationss for cell lysis of human lung fibroblasts after infection with M. genitalium (18) and celll disruption after invasion with hi. penetrans (16). The intracellular localisation can protect mycoplasmass against the host immune system and antibiotics and may be responsible for latentt or chronic infection states. M.M. pneumoniae has been shown to parasitize cell surfaces of mammalian cells and to enter thee intracellular environment where it is located throughout the cytoplasm and perinuclear regions.. It can persist intracellularly for at least 7 days (19). Baseman isolated M. pneumoniae mutants,, which are able to cytadhere but not to invade cells, suggesting separate mechanisms forr adherence and invasion (19).
10 0
Introduction Introduction
2.2. Epidemiology 2.11 Epidemiology M.M. pneumoniae causes usually a mild respiratory disease. Most cases will not lead to a doctor'ss visit and confirmed diagnosis is often not accomplished in daily practice. Available dataa on morbidity and mortality due to M. pneumoniae
infection from regular sources
(statisticss of death due to pneumonia, statistics on hospitalisation due to pneumonia or laboratoryy data on M. pneumoniae infection) thus will reveal only a minor part of actually occurringg M. pneumoniae infections. Thee way diagnosis of a M pneumoniae infection is established will influence the outcome off epidemiological studies. Most studies on M. pneumoniae epidemiology performed in the past,, made use of data obtained by serological testing or by culture of the organism. A deficientt knowledge in parameters influencing the antibody response exists. Persistent antibodiess and antibody response after reinfection may differ and probably depend on the age andd immune status of the people under investigation. Although isolation of M. pneumoniae is tediouss and takes several weeks to complete, culture of M. pneumoniae
has provided
additionall information for epidemiology of the organism only in settings in which special attentionn to culture was paid. Prolonged shedding of the micro-organism has been described in certainn populations and is probably related to several factors like the immune status of the host,, the age of the host, intervention with antibiotics, and may be, the virulence of the strain. However,, the asymptomatic carrier state is believed to be rare, although contradictionary resultss have been reported (20,21). Manyy studies have been performed in outbreak investigations of respiratory disease due to M.M. pneumoniae, for example among military and in boarding schools (22-24). Long term studies,, as performed in civilian populations in Seattle (1962-1975)(25) and Tecumseh (26), havee provided data on community infection rates and epidemiological patterns. M.M. pneumoniae infection occurs in temperate climates throughout the year, but the absolute numberr of infections is higher during the winter season. Because most respiratory infections duee to other pathogens such as rhinovirus, respiratory syncytial virus and influenzavirus occur inn wintertime, the proportion of M. pneumoniae infection among respiratory infections is highestt in summer. Althoughh M. pneumoniae is endemic in most civilian populations where the infection has beenn sought for, an epidemic pattern is evident from long-term population studies (27-32). Epidemicss occur with intervals of 4-5 years. However, a change in this epidemic pattern has beenn observed in Denmark, possibly related to a change of protective immunity in children, whichh can be a result from increased use of day-care facilities (27). As can be concluded from longg term population studies among patients with pneumonia (25) and data from laboratories registeringg M. pneumoniae
infection (29)(this thesis), the more severe M. pneumoniae
11 1
ChapterChapter 1
infections,, occur predominantly in school-age children, with another incidence peak in young adults.. Although these data suggest that the overall incidence is lowest in the over 40 age group,, it is likely that there is an underestimation of M. pneumoniae infection occurring in peoplee older than 40 years. Antibody titers due to M. pneumoniae infection in older people are significantlyy lower compared to younger people (29; this thesis, Chapter 3), implying that M.
pneumoniaepneumoniae infection in the older age groups is not diagnosed in case only serological assays aree performed. In studies performed among outpatient populations with respiratory tract infection,, in which diagnosis of M. pneumoniae infection was made by PCR, no difference in incidencee between the different age groups is found (33; this thesis, Chapter 5). 2.22 Results of registration o/M. pneumoniae from Laboratory Reporting Systems in Europe Too gain insight into the epidemiological pattern of M. pneumoniae in Europe, the data from nationall diagnostic laboratory reporting systems in different countries have been analysed. Onlyy countries having data reported per quarter or per month for at least 10 years were included.. These countries were: Finland (1972-1998)(34), England and Wales (19751998X35),, the Netherlands (1981-1998)(36) and Denmark (1972-1999)(37). Data from Icelandd (1987-1996) reported earlier (29), were also included in the analysis. As M. pneumoniaepneumoniae
infection is not a notifiable disease, the information available is based on
volunteerr reporting systems (Finland, England/Wales, the Netherlands) or on data from the nationall laboratory performing M. pneumoniae tests (Denmark, Iceland). The reported data obtainedd from laboratory diagnosed M. pneumoniae
infection, most often by antibody
detectionn in serum (CFT and/or EUSA), are presented as absolute numbers. Inn those countries for which monthly data were available, most infections occurred during thee winter period (November until March)(Fig 1). In epidemic episodes, however, the infectionn is diagnosed during the whole year, without preference for the winter period (Fig 2). Thee data showed that endemic cases occur on a regular basis with epidemic outbreaks every 4 too 5 years. This pattern is most regular in England/Wales and possibly in Iceland, for which countryy only 10 years data are available. Epidemic outbreaks in the different countries do not alwayss coincide (Fig 2), most likely because of the protective immunity built up in a certain population. . Thee laboratory reporting systems from England/Wales and the Netherlands also register sex andd age of the patients diagnosed with M. pneumoniae. M. pneumoniae infection occurred slightlyy more frequent in male patients with an exception for the 16 to 40 years age group in whichh more female patients were diagnosed with M, pneumoniae infection (Fig 3). In both countriess there was a peak incidence of infections in young children (4 to 10 years) and a smaller,, second peak in adults of 30 to 40 years old (Fig 4).
12 2
Introduction Introduction
England/Wales s (1975-1998) ) 3000 0 2500 0 2000 0 1500 0 1000 0 500 0 00 44 week periods
Thee Netherlands (1981-1999) ) 1200 0 1000 0 800 0 600 0 400 0 200 0 55
00 jann
feb mrch apr
may June July
aug sept
oct
nov
dec
aug sept
oct
nov
dec
nov
dec
Finland d (1972-1999) ) 1600 1400 1200 1000 800 600 400 200 0
0 0 0 0 0 0 0 0 0 jann
feb mrch apr
may June July
Denmark k (1990-1999) ) 2000 0 1500 0 1000 0 500 0
l.n.n.n.nn n I:
00 jann
II m I m I HM I n a I i leb mrch apr may June July aug sept
oct
Figuree 1 Seasonall distribution of M. pneumoniae in 4 Northern European Countries (data derived from (34-37))
13 3
ChapterChapter I
MpMp reports &igland/Wales (per month) (n=24,239) ) 400 0
755
77
79
81
83
85
87
89
91
93
95
97
Mpp reports the Netherlands (per month) (n=9672) ) 250 0 200 0
150 0 100 0
vlhMu^li^^ ^
50 0
811 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 MpMp reports Finland (per month) (n=12,105) )
722 74 76
78 80 82 84
86 88 90 92
94 96 98
MpMp reports Denmark (per quarter) (n=29,333) )
1500 0
833
86
89
92
95
98
MpMp reports beland (per quarter) (n=762) ) 60 0 40 0 30 0 20 0 10 0
iMii lAiiili 93 3
Figuree 2 Endemicc and epidemic periods of M. pneumoniae as registered by clinical diagnostic Northernn European countries (data derived from(29;34-37))
14 4
laboratories in 5
Introduction Introduction
Agee and Sex distribution rVfc patients England/Waless 1990-98
(n=12,943) ) 2500 0 2000 0 1500 0
number r 1000 0 500 0
AA AA II I 0-44
II male II female
5-15 16-25 26-40 41-60 > 60 agee category
Agee and Sex distribution N^> patients thee Netherlands 1986-94 (n=4892) ) 1000 0
fa fa
11 11 1
800 0
600 0 number r 400 0
r?fl
jj j
200 0
00
0-44
I—HL
Ell male female
IJIJtlLBi i 5-15 16-25 26-40 41-60 >60 agee category
Figuree 3 Agee and sex distribution of patients diagnosed with M. pneumoniae Netherlandss (data derived from (35;36))
infection in England/Wales and the
15 5
ChapterChapter I
Mpp infections by age (England/Wales 75-98) (n=28,609) )
IlllllllMlMUl l 00
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 age e
Mpp infections by age (the Netherlands 81-96) (n=7020) )
II
O OO
number r
4000
100 0
III I lilllllllllllllillllllllllllllllllliii
..i,inil,in....,i,l.iM.
33 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 age e
Figuree 4 Agee distribution of patients diagnosed with M. pneumoniae infection in England/Wales and the Netherlands (data derivedd from (35;36))
16 6
Introduction Introduction
2.33 Transmission Transmissionn of respiratory pathogens can occur by aerosols (e.g. influenza virus, BordetellaBordetella spp.), by personal contact (e.g. Epstein-Barr virus) or even via contaminated objectss (respiratory syncytial virus). In the case of M. pneumoniae it is uncertain whether the spreadd is primarily by droplets, or by direct or indirect contact, or by all of these three routes. Infectionn with M. pneumoniae with small-particles (2-3um) in the hamster model resulted in bothh upper and lower respiratory tract infection, whereas infection after exposure to largeparticlee aerosols was limited to the upper respiratory tract (38). Thee incubation period of M. pneumoniae is approximately 3 weeks. Spread of a M. pneumoniaepneumoniae
infection
in
the community
is slow.
However,
within
communities,
microepidemicss can arise, in which spread probably occurs from person to person. Only a few commonn source outbreaks of M. pneumoniae have been reported (39-41).
3.. Clinical disease 3.11 Respiratory infection M.M. pneumoniae infection should be suspected in patients with a respiratory infection with clinicall symptoms as seen with influenza-like illnesses such as fever, cough, headache, malaise,, and myalgia although the onset of an infection with M. pneumoniae is usually less abruptt compared to an influenza virus infection. Also in people with milder respiratory infectionss M. pneumoniae may be the cause of such a disease. Non-productive cough is a characteristicc sign in M. pneumoniae infection. In children cough can be paroxysmal. In an estimatedd 5-10% of patients M. pneumoniae infection progresses to tracheobronchitis or pneumonia.. Sputum may be produced in such cases. Gram staining of sputum specimens revealss only inflammatory cells. Pneumonia is usually mild and self-limited, resolving even withoutt antibiotic treatment (42). However, severe and fulminant cases have been reported (43,44)) and may be underdiagnosed. 3.22 Complications AA variety of complications of M. pneumoniae infection have been described, but their frequenciess are rarely known (45). Skin rashes, one of the commonest complications occurred inn 10-20% of the recognised M. pneumoniae infections (46,47). The Stevens Johnson syndromee (erythema multiforme) although rare, can be a life threatening complication. Other non-pulmonaryy common complications comprise haemolytic anaemia, thrombocytopenia, thrombosiss
and
disseminated
intra-vasculair
coagulation.
Furthermore,
neurological
complicationss like encephalitis, meningitis and the Guillain Barre syndrome, but also myocarditiss and arthritis have been described and are believed to be due to circulating
17 7
ChapterChapter 1
immunee complexes. Recovery of the organism from cerebrospinal fluid (48,49), synovial fluid (50-52)) and the description of mycoplasmal bacteremia as detected by molecular methods (53,54)) suggest systemic spread of the bacterium.
4.. The bacterium and the host 4.11 Virulence and evasion of the host immune response Thee clinical picture of mycoplasma infections is more suggestive of damage due to host immunee and inflammatory responses rather than to direct effects by mycoplasma cell components.. M. pneumoniae
can be considered as a surface parasite of the respiratory
epithelium.. The adhesion is essential for colonisation and infection as the loss of adhesive capacityy results in loss of infectivity. For M. pneumoniae and M. genitalium the mycoplasma adhesins,, their genes (PI, P30, and MgPa) and encoded proteins have been studied most extensively.. The topography of the adhesion molecules with epitope mapping using monoclonall antibodies against adhesion molecules in situ and against synthetic oligopeptides providedd insight into the conformation of the adhesins in the membrane (55). However, in M.
pneumoniaepneumoniae a number of accessory membrane proteins are involved in the process of gliding motilityy and concentration of the adhesin molecules at the attachment tip organelle. Some of thee genes and encoded proteins of the M. pneumoniae cytoskeleton have been identified (30, 400 & 90 kDa, P65 and HMW1 and HMW3) and are shown to be proline rich and possess otherr characteristics of eukaryotic cytoskeletal proteins (56-59). Forr persistence in the host, pathogenic bacteria develop mechanisms to deal with the host immunee response. One of these mechanisms consists of variation in antigenic structure in orderr to avoid host immune recognition. For M. hominis deletions in a repeat-containing gene encodingg a surface localised antigen resulted in antigenic variation (60). Also in the case of M. genitaliumgenitalium regions in the MgPa adhesin gene encoding adherence mediating epitopes have beenn described to be highly variable, thus leading to avoidance of the host immune response (61).. For M. pneumoniae, deletion of repeated sequences in the gene encoding the 30 kDa proteinn of the M, pneumoniae attachment organelle, resulted in a hemadsorption negative mutantt of M. pneumoniae (62). Regions of the PI cytadherence gene which are recognised by cytadherence-inhibitingg anti PI monoclonal antibodies have been shown to vary, at least betweenn PI group I and II M. pneumoniae isolates (63,64). 4.22 Host susceptibility to M. pneumoniae infection Peoplee with congenital or acquired immunodeficiencies are recognised to be more susceptiblee for a variety of mycoplasma infections. The number of recurrences of M.
pneumoniaepneumoniae infection in such patients compared to people with normal immunity is also
18 8
Introduction Introduction
higher.. In hypogammaglobulinaemic patients and in patients undergoing organ transplantation andd immunosuppressive therapy, dissemination of mycoplasmas such as M. pneumoniae, M. hominishominis and Ureaplasma urealyticum to joint and bone tissue has been reported (50,65-69). Primaryy infection with M. pneumoniae does not protect against a subsequent infection with thee bacterium as follows from clinical studies (70) and animal studies (71). Reinfected animalss show more lymphocyte infiltration than primary infected animals, indicating that specificc cell-mediated host response might play a role in the pathology of a reinfection. Experimentall reinfections of mice showed elevated mRNA expressions for proinflammatory cytokiness (72). In humans, the difference in clinical disease between a mild primary infection andd more severe manifestations after a second infection with M. pneumoniae (73), might be duee to enhanced immune response as found in the reinfected animals (74).
5.. Diagnosis Diagnosiss of M. pneumoniae infection relies mainly on laboratory tests, as discrimination off M pneumoniae from other (mostly viral) respiratory pathogens on clinical parameters only, iss difficult (75). Specific laboratory diagnosis can be made directly, by culture or detection of antigenn or DNA of the organism, or indirectly by detection of antibodies. 5.11 Culture Culturee of M. pneumoniae is labour intensive and requires specific media and experienced personnell to perform. M. pneumoniae is a relatively slow growing bacterium. After adaptation too artificial media the mean generation time is 6 hours (76). In clinical practice this means that incubationn from 1 week to 4 weeks is necessary for propagation of the micro-organism, which makess culture inappropriate for the routine diagnosis in a clinical setting. If culture is attempted,, the transport medium should contain peptide broth, serum albumin and antibiotics too retard overgrowth by other bacteria. Throat swabs, nasopharyngeal aspirates and sputum sampless are all suitable for culturing of M. pneumoniae, provided they have been suspended in appropriatee transport medium. Inoculation should be performed within 24 h or, if not possible, sampless should be stored at -70 °C. Culture media to be used for M pneumoniae have to containn beef heart infusion broth, supplemented with fresh yeast extract and horse serum providingg sterols and nucleic acid precursors. Inoculation is performed using both solid and liquidd medium, the latter containing glucose and phenol red. M. pneumoniae
ferments
glucose,, which is detected by a colour change of the medium from red into yellow. On solid mediaa M. pneumoniae appears in 'mulberry' colonies (Fig 5), which are able to hemadsorb chickk and guinea pig erythrocytes. Specific antisera are required to provide definite identification,, although close serologic relationship between M. pneumoniae
and M.
19 9
ChapterChapter I
genitaliumgenitalium may encounter difficulties in identification (77). Although the usefulness of culturingg M. pneumoniae in daily practice is limited, culture remains important as a standard inn studies in which non-cultural methods are evaluated. Also the development of antimicrobial drugg resistance and antigenic variation can only be studied if clinical isolates of M. pneumoniaepneumoniae are available.
Figuree 5 MM pneumoniae colonies on agar (40x magnification) (courtesy to AF Angulo, RIVM Bilthoven, The Netherlands) )
5.22 Direct detection of M. pneumoniae Ass an alternative for culture, direct antigen tests have been developed. A species specific probee test (Gene-Probe), an EIA and immunoblot assays making use of monoclonal antibodies againstt M. pneumoniae proteins have been applied in direct detection of (antigen of) the micro-organismm in nasopharyngeal specimens (78,79). Inn the last decade studies using amplification of species specific fragments of the M.
pneumoniaepneumoniae genome by PCR have been published. Target sequences used for molecula detectionn of M. pneumoniae are mostly chosen in the PI cytadhesin gene (80-85) or in the 16 SrRNAA gene (86,87). Forr preparation of clinical samples before amplification, various procedures have been described.. Since mycoplasmas lack bacterial cell wall material, DNA can be made available byy relative simple procedures. A boil-freeze method (thermal shock) has been applied to nasopharyngeall aspirates (85) and also lysis with proteinase K on aspirates (88), bronchoalveolarr lavages (84), and throat swab specimens (75,89) has been applied. For preparationn of tissue or blood specimens a more elaborate DNA extraction is required. The
20 0
Introduction Introduction
usee of an internal process control is necessary in order to avoid false negative results (82,85,90).. Up to 20% of the throat swab samples after a proteinase K lysis showed inhibition off the amplification process (90) and up to 25% of nasopharyngeal aspirates after freezeboilingg showed inhibition of the PCR (85), as detected by the lack of the internal control band afterr gelelectrophoresis. Using 6 globine primers, a more roughly way to test for inhibition, up too 10% of the aspirates showed inhibition after proteinase K lysis (88). Identification of the PCRR products has been performed by (microliter well) hybridisation (89,91) as well as by a nestedd PCR (90). Detection limits of M. pneumoniae by PCR in clinical samples have been describedd to vary between 1.0-50 colony forming unit (CFU), 10-100 colour changing units (CCU)) and 5-50 fg of template DNA (53,82,87,90,92). Only a few studies compared reproducibilityy of PCR results obtained with different primersets in different laboratories (75,85). . 5.33 Serology Inn routine laboratories antibody detection in serum is the most commonly used technique in diagnosiss of M. pneumoniae infection. The first serological assay performed was the cold hemagglutininn (CA) test. Cold agglutinins are non-specific IgM class antibodies against the I antigenn of erythrocytes. The specificity of the test can be increased when a cut-off titre of >40 iss used as diagnostic criterion (93). As more specific tests became available, the CA test is not commonlyy used anymore. Thee most widely used serological assay is the complement fixation test (CFT) in which CF antibodiess against M. pneumoniae, in conjunction with antibodies against other, mostly viral, respiratoryy pathogens are determined. The sensitivity of this assay depends on whether the firstt serum sample is collected early or late after onset of illness and on the availability of pairedd sera collected with an interval of 2 to 3 weeks. On interpretation of positive CF titers onee has to bear in mind that the membrane glycolipids-based antigen from M. pneumoniae is notnot highly specific. Cross reactivity with e.g. vegetable lipids (94) and human brain tissue antigenss has been demonstrated (95). ELISAA tests have been developed to detect specific immunoglobulins of different classes, mostt of them using a crude M. pneumoniae
antigen, which includes the cross reacting
glycolipidd fraction. More specific test results can be obtained by using isolated PI protein as antigen,, which is one of the major virulence factors of M. pneumoniae (96). Proteins derived fromm parts of the PI gene have also been applied as antigen in an ELISA test (97). Commerciall availability of these tests sofar is limited. Immunoblotting, to confirm positive resultss from the less specific serological assays, have shown strong reactivity against the 169 kDaa PI protein of M. pneumoniae (98), but reactivity against this protein among clinically healthyy persons interferes with conclusive interpretation. Recently, a commercially available
21 1
ChapterChapter I
immunoblott making use of various recombinant proteins based on M. pneumoniae genome sequencee data (3) has been developed.
6.. Treatment and prevention 6.11 Treatment Upperr respiratory tract infection due to M. pneumoniae is mostly undiagnosed and is not treatedd with antibiotics. Also pneumonia is often a self-limiting disease, but appropriate antimicrobiall treatment shortens the duration of the illness (99; 100). Respiratory infection causedd by M. pneumoniae
is generally treated with tetracycline or erythromycin. As
mycoplasmass lack a cell wall, M. pneumoniae is unaffected by B-lactam antibiotics such as penicillinn
and cephalosporins. Clinical
improvement
upon institution of
appropriate
antimicrobiall therapy is not always accompanied by early eradication of the organism from the respiratoryy tract. The capacity of M. pneumoniae to reside intracellular^ may be responsible forr this. Also the newer macrolides (clarithromycin and azithromycin) and to lesser extent fluoroquinoloness have been found to have in vitro and in vivo activity against M. pneumoniae (101). . 6.22 Prevention Inn terms of transmission, the respiratory route is the least subject to effective control of infectiouss diseases. In non-respiratory transmission such as vehicle-borne, gastro-intestinal infections,, intervention is possible in the food chain or in drinking water systems (102; 103). Inn case of vector borne infections the vector can be eliminated (104) and in case of genital infectionss change in behaviour will reduce transmission of the causative micro-organism (105).. For some respiratory pathogens control is possible by intervention in the environment. Thiss is the case for example with Legionella pneumophila (106) or Chlamydia psittaci (107). However,, for many respiratory pathogens, including M. pneumoniae, environmental control is notnot feasible and the only way of controlling these infections is through immunisation of the hostt or providing chemoprofylaxis. Preventionn of M. pneumoniae infection through vaccination has been studied as early as the 1960ies,, the time that M. pneumoniae was recognised as a respiratory pathogen. These early vaccinee studies made use of an inactivated M. pneumoniae vaccine after administration of whichh volunteers were challenged with agar-grown
mycoplasma. Those
responding
serologicallyy to the vaccination showed protection to respiratory infection. In volunteers who failedd to respond to the vaccine with antibody development, more severe disease developed afterr challenge compared with the controls (108). In larger vaccine trials among military personnell this sensitisation effect could not be confirmed and the protective efficacy of the
22 2
Introduction Introduction
vaccinee against development of M. pneumoniae pneumonia was about 66% in the first year followingg vaccination (109). However, the overall success of formalin inactivated vaccines hass been disappointing and no acceptable live vaccine has been developed for human use. Thee molecular characterisation of the major adhesin PI of M. pneumoniae, prompted trials too use purified PI as vaccinogen. The results of immunisation with PI in animal studies were howeverr disappointing (110), showing the same lymphocyte infiltration in lungtissue after challengee with M. pneumoniae in immunised animals compared to control animals. The conclusionn is that until now, no useful vaccine (inactivated or live) for M. pneumoniae has beenn developed. DNA vaccines, having the advantage of inducing both humoral and cell mediatedd response, have been applied successfully in animals preventing mycoplasmal disease (M.(M. pulmonis, M. hyopneumoniae) (111). This generation of vaccines may become useful in humann mycoplasma disease in the future as well. Onlyy limited data is available on the effect of prophylactic antibiotic use. In a long-term caree facility prophylactic antibiotic usage has shown to significantly reduce the secondary attackk rate of M. pneumoniae infection (112).
23 3
ChapterChapter I
Scopee of the thesis
MycoplasmaMycoplasma pneumoniae is a common respiratory pathogen in humans. The usual mild clinicall presentation of the infected patient does not encourage laboratory diagnosis to be performed.. However, in case antibiotic treatment is instituted this should be different from the firstt choice recommended antibiotics for respiratory infections in general practitioner (GP) settings.. As the organism is notorious for its difficult laboratory diagnosis, molecular techniquess should enable fast diagnosis and subsequently appropriate antibiotic treatment. Currentt knowledge of the epidemiology of M. pneumoniae is based on data predominantly derivedd from studies using serology or culture for diagnosis. Inn this thesis the value of molecular techniques in diagnosis of M. pneumoniae in both childrenn and adult patients with a respiratory infection is investigated (Chapters 2 and 3). For reliablee detection of M. pneumoniae DNA by PCR, control for inhibition of the amplification reactionn is required. Therefore, we developed an amplification control (AC) to detect inhibition,, which is described in chapter 4, This AC was applied to clinical specimens, submittedd for M. pneumoniae PCR. Furthermore, we applied a semiquantitative PCR to enablee discrimination of strong and weak positive PCR results in specimens obtained from hospitalisedd and non-hospitalised patients with a respiratory tract infection (Chapter 4). Too expand knowledge of epidemiological patterns of M. pneumoniae, the PCR was applied too nose/throat specimens obtained from patients with acute respiratory infection, identified by GPss in a routine surveillance for respiratory pathogens. M pneumoniae transmission was studiedd in the households of the M, pneumoniae positive patients (Chapter 5). Molecularr epidemiology of M. pneumoniae is hampered, as variation in its genome allows thee distinction of only two types. In order to refine molecular typing, clinical isolates of M.
pneumoniaepneumoniae collected over time in Denmark and the Netherlands, were subjected to various molecularr typing methods (Chapter 6). For identification of targets, which should enable directt genotyping of M. pneumoniae in clinical specimens, PI genes were sequenced. In addition,, these sequences were analysed to uncover possible mechanisms generating PI gene sequencee variation (Chapter 7).
24 4
Introduction Introduction
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