Epidemiology, Risk Factors and Treatments for Anti ...

Review Article

Digestive Diseases L. v McForlrrnd Deportment of Medicinal Chemistry, school of Pharmacy, University of Washington and Biocodex, Inc., Seatile. wash.. USA

Epidemiology, Risk Factors and Treatments for Anti biotic-Associated Diarrhea

................................................... ...................,....................................................................................... Key Words

Abstract

Antibiotic complications Antibiotic-associated diarrhea Clostridiunz difficile Epidemiology Diarrhea, risk factors

Antibiotic-associated diarrhea (AAD) is a conimon complication of antibiotics and recent findings on tlie epidemiology, etiologies and treatment strategies are reviewed. Rates of A A D vary Crom 5 to 39% depending upon the specifictypcoCantibiotic. Theseverity of AAD may include uncomplicated diarrhea,colitisorpseudoiilembranouscolitis.The pathogenesisofAADlliay be mediated through the disruption of the normal flora and ovcrgrowth of pathogens or through metabolic imbalances. The impact of A A D is reflected by increased hospital stays. higlierinedicalcostsand increased ratesofcomorbidity. The key to decreasing these consequences is prompt diagnosis followed by erective treatment and i~istitutionof control measures.

Introduction

Antibiotics remain tlie most effective available weapon in the battle wiht infectious diseases. but thcir use is not without co~nplications.Currently, the go21ls in the development of new antibiotics include finding efrective antibiotics with a broad spectrum of action, while at the same time developing strategies to co~llrolantibioticresistance and limitiiig complications associated with antibiotic use [I, 21. One of the most common complications arises whcn antibiotics disrupt tlie ecology of normal intestinal microflora, which results in antibioticassociated diarrhea (AAD). A A D has bccn increasingly reported in a wide variety of paticnt populations including orthopedic patients [3]. obstetricigynecological patients [4], general medicine patients [5-81, nursing liotnes [9, 101,patients with AIDS [I I] andcancer [I21 or ambulatory patients [13]. A A D may be divided into two in21.ior types: uncomplicated diarrhea (not associated with ('1o.stridi~ondificile) and C. rl;lji'cilc disease (diarrhea. colitis or pseudomeinbranous colitis). Thc objective of this re-

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view is t o summarize the epidemiology and risk factors for A A D and suggest recorninendations for treatment and control.

History

AAD became a recognized clillical concern in the 1950s when the use of broad-spectrum antibiotics (tetracycline and chloramphenicol) increased. However, little attention was given to this seemingly benign complication until tlie frequency o f a relatively rare but serious disease, pseudomembranous colitis (PMC). was reported in 10% of patients receiving clindaniycin [14]. Attempts to uncover the etiologic agent remained elusive until 19771978 when tlie link between A A D and an opportunistic anaerobe, C rlif%-'ciiewas discovered [15-181. In thc 1980s. much of the attention centered on nosocomial outbreaks that were due to dificile, even though diffii.ilr2 was only implicated in about one quarter of thc outbreaks. An extraordinary amount of research suc-

Table 1. Frequency of antibiotic-icraociated di;ll.~.Iiciland ('. il;/ficiicik ;~ntibiotic-associated diarrhea in defined populations

Patient population AAD in non-outbl-r;tk situations Febrile %ranulocytopcnic patients Adults in community lhospiti~l Adults with respir;itory or intestinal infcclioos Communily lhospival pittients

Country

Number in study Frequency'

Reference

USA USA France

136 64 388

Winston et ;iI. (531 Surdwicz et zil. [63] Ada111 et al. [48]

USA

96

USA Spain

.?99 386 26 1 240 110 140 I SO

291 100 22100 181100

McFarland el al. [5]

- .

-

CDAAD Outbreak: gencrsl medicine ward Outbratk: adult patients Adults Adults on infectious disease wlrd Piilients given p-lactams Patients given clindamycin Adult sulgcry patients with prophylactic antihiolics Children s 5 yrars old Adults in tel-tiilly =ire hospital Adults on OBICYN \el-iricc --

Oulp;!ticnt populations Adults in H M O

Fritnce

Francc Thailand Thailand Unitcd Kingdom India USA USA USA

-

-

~

LISA

Ill NA NA 74.120

--

662.5001 person-yealr

-

McFal.land and Stamm 11511 Knobel el al. 1641 DeBarbeyac et a 1 [49] Hulin el al. [52] Thtlmlikitkul el al. [I521 Thamlikitkul ct ;!I. [I521 Bulstrode el al. [1 131 3.6'%, 2.21100 2.21l00 0.02'%

Dutta et al. [50] Brown et al. [27] Lai el sl. [I 191 James el al. [4]

-

-

7.71100.000 Hirsclihorn el al. [54] person-)cars

AAD=Aotibiotic-i~ssociateddiarrhc;~;CDAAD= C ~ l ~ f h rAAD: i i ~ ~ N 4 = d a t a not auailable: HMO=heziltli maintelrencc orsanization: OBIGYN=ohsletl-ics and eynccolopy. I Freqitcncy is hy incidence if given ;IS ;i vale or by prevt~lcnceif given as a percentage.

cessfully documented the populations at risk, routes of transmission, methods which enabled the documentation of the spread of specific strains during outbreaks, mechanisms of action for the toxins o l ' C diflicilc and treatment strategies [19-261. Other possible etiologies of A A D (Conclidn, E?~rerococc~o,/uecoiis) were also being explored, but the research was not as productive. This past decade has brought greater understanding of the risk factors for AAD, mechanisms, other possible etiologies, diagnostic assays, treatment strategies and methods of control [27-351. Although much has been discovered about AAD, it continues to persist, especially with the development of newer broad-spectrum antibiotics. A A D has been shown to be a risk for the individual (in whom A A D may lead to sc~iouscomplications). hut also as a risk for the medical community (reflected by the increased frequency of hospital outbreaks). As current treatment regimes are not always successful. A A D will be acontinuing medical concern into thc next millennium.

The reporled incidence of A A D ranges from 0.44 to 261100 (table 1 ) depending upon host factors (age, health status. etc.), hospitalizatioil status and the presence of a nosocomial outbl-eak. The highest frequency of A A D is found during l~osocomialoutbl-eaks when cases are clustered by time. exposure, susceptibility and proximity. Hospital outbreaks of A A D are to be expected when an inciting agent (antibiotics), a n iilfectious agent and a susccptible patient population are intermixed. Most. if not all, of the reported nosoconiial outbreaks of AAD are due to C d;ficile; but this is not to collclude that thcre are no outbreaks of A A D that are caused by other etiologies. C. ~l#icilt~is the only etiology that has available tools (strain identification, typing techniques, culture assays, environmental sampliiig techniques) that are widely av;iilablc [3h. 371. Numerous outbreaks of C d~ficileassociated A A D (CDAAD) have been reported interna-

Rcview of AAD

Oit. n i ~ 11)98:16:292 307

Frequency of AAD

293

tionally since the 1980s with the number ofinfccted cases ranging from 15 t o 78 patients during outbreaks [3X4I]. Early studies of nosocomial outbreaks of CDAAD successfully documented that this organism could be transmitted by roommates, hospital personnel and indirectly transmitted on environmental foniites resulting in new infections [26. 42, 431. Outbreaks of non-C. diJfiriL etiologies probably d o exist and may be documented when diagnostic tools bccome available. Unfort~ulately,prospective studies of ~iosocomialoutbreakso~AADnotassociated with C. drficile have not been reported. Another problem with documenting nosocomial outbreaks of A A D is that diarrhea is a common symptom in hospitals and not all the outbreaks of diarrhea are associated with antibiotic exposure 1441. Nosoconiial outbreaks of diarrhea have been reported due to rotaviral infections 145.461, calicivirus [47], and Entarttoebct hisiolytica [44], but their association with a~itibioticsis unclcar. Prcvalence of A A D and CDAAD during non-outbreak periods (endemic) in patients may range from 0.5 to 29% 17, 48-52]. Winston et al. 1531 studied 429 febrile granulocytopenic patients receiving antibiotics and found that 29% receiving cefoperazotie plus piperacilli~i developed AAD. In 96 pdticnts receiving 0-lactam antibiotics at a community hospital. 14 (14.6%) developed A A D 151. The incidence of CDAAD in general medicine inpatients in the absence of a dcfined outbreak of C ~l$j5cilealso averages 15-201100 patients. Olson et al. [7] studied 166,952 hospital admissions over a 10-year pcriod (1982-1991) and found a prevalence of CDAAD of 0.5'%,. DeBarbeyrac ct al. [49] found 401261 (15.3'%,) patielits in a French hospital developed CDAAD. Hutin et al. [52] studied 240 patients admitted to an inrectious disease ward and found 13.3'!4 developed CDAAD. The lowest frequency of A A D is usually observed in nonhospitalized. ambulatory patients. Hirschhorn et al. [54] studied members in a health maintenance organizatioliandfound7.7per 100,000perso11-yearsofcommunityacquired CDAAD. Lower rates observed in outpatients may be due to their generally higher health status than hospitalized patients and also t o the lack of exposure to nosoconiial pathogens that commonly contaminate hospital environments.

Mechanisms of Pathogenesis

The pathogenesis of A A D involves two ~ i ~ a j mechaor nisms that are initiated by the disruption of normal intes-

294

Dig Dis 1998;16:29?-307

tinal flora: over_prowtli by opportunistic pathogens o r alterations of metabolic functions of the flora. Colonization resistance, or the ability of the normal flora to resist overgrowth by pathogenic organisms. has been well documcnted [55. 561. Cololliratioll resistance operates on sevel-al levels within the intestinal lumen: normal flora competes for microbial nutrients, produces bacteriocilis or toxin-degrading proteases and sheltcrs pathogen attachment sites o r toxin receptor sites 138, 561. Colonization resistance is a result of the interactions of numerous specics offlora and the ~io~ispecific destruction by broadspectrum antibiotics results in the greatest impact on the flora. Difcrent types of a~~tibiotics have varying rates of AAD, which may be due to two factors: the spectrum of activity against the normal flora and the degrec of absorption from the intestinal tract. Narrow-spectrum antibiotics have low rates of A A D and broad-spectrum antibiotics, especially those effecting anaerobic normal flora, are associated with hisher rates of A A D 1571. The degree of absorption has also been found to be influential in the rates of AAD. Antibiotics that are poorly absorbed from thecololl o r are secreted in bile (such asclindamycin, cefixitne, ceftriaxone o r cefoperatzone) are associated with high rates of A A D [%I. Antibiotics with high absorption such as doxycycline and cefaclor havc lower rates of AAD 159, 601. Once colonization resistance has been disrupted, overgrowth by opportunistic pathogcns may occur. Several pathogens have been studied that may be capable of this opportunistic overgrowth and the supporting evidence for their role in A A D is given below. CIostri~liun7d1Jfici1r The majority of studies involving A A D havc focused on CDAAD as this pathogen came t o the attention of thc medical community when the frequency of nosocomial outbreaks began to increase. Approximately 26-50'h of A A D cases and nearly all (95-99%) cases ofpseudometnbranous colitis can be attributed to C. ~liflicile[5, 61-64]. The effect of overgrowth by C. d;ficile results in toxinmediated pathogenesis due to two large heat-labile toxins called toxinA and toxin B. Toxin A i s a potent el~lerotoxin and weakly cytotoxic. Toxin B is a potent cytotoxin and, until recently, was not considered a potent enterotoxin. Riegler et al. 1651 found that toxin B was more potent than toxin A in damaging human colonic epitheliunl in vitro and may thus play an important role in virulence. Toxin A has been found a potent stimuli of human neutrophil IL-8 production. and toxin A may also stimulate neutrophils to increase adherence to fibrino_een-coated surfaces 1661. The efTects of these two toxins on the muco-

McFarli~nd

sal cells is to act (via Rho protein) on thc cytoskelelon. disrupting the f-actin. distorting the celluler shape and broadening the tight junction space bctween the cells. These changes lead to fluid loss and diarrhea [67]. In addition t o the physical disruption of enterocyres, toxins A and B attract polymorphonuclear leukocytes and otlier inflammatory cells to tlie site which can be observed ; ~ t endoscopic or histologic examination [6X. 691. Although C rlrficile appears to cause from 25 to 33'%,of the cases of AAD and has bcen the most thoroughly studied. othcr possible etiologies need to be examined to fi~rtlierunderstand AAD.

~ x b b iintestinal t loop models [75]. The cytotoxin. isolated from a patient with K. 0.1-yroccr antibiotic-associated hemorrhagic colitis, was injected into ligated ileal and colonic loops and caused a dose-dependent accumulation of bloody fluid in the ileal loops. but not the colonic loops. This possible etiology requires fnrtlier investigation beforc its rolc in A A D is determined. S ~ ~ r ~ ~ l r ~ ~ / o ca~rrsu.s cocrr.s

Historic:~lly. this bacteria was once implicated as the major etiologic agent of dial-rhea. but subsequent studies on AAD caused by clindaniycin (an effective anti-staphylococcal antibiotic) discounted this theory [57]. The discovery of C cl#~cil~,as an etiologic agent for AAD turned Condido olhicans The role of C olhicill7.~in A A D has bcen investigated tlie attention ;way from this organism until recently. Sevsince 1995 with differing conclusions [70]. Fir111 COLICIUeral reports from Japan have reported an association of sions have been clouded by the presence of C: nN>ic(rn.~ AAD and ilifectio~iwitli methicillin-resistant S oureu.r in normal healthy populations and he lack of biopsy [76-781. These patients were neeativc for stool carriage of evidence of patliogenesis [57, 711. Levine ct al. [70] re- C i l i f i c i l ~or other enter-ic palllogens. Cases o f A A D assoviewed studies of patients with diarrhea who also had ciatcd with methicillin-resistant S. nui.r.lrs liave not been positive cultures for C u l i ~ i c r mand . ~ found 2051224 (911%,) I-eportcd outside Japan. but it may be an area of concern. ofpatients treated ~ : i t horal nystatin repol-ted the iiiarl-Ilea The inducing antibiotics for S ar~reci.sA A D are pri~niarily improved o r completely resolved. Hou:c\.el: in this sti~dy tetracyclines. chlornmphenicol and neomycin and the panot all tlie patients had prior antibiotic exposure and of thology is evident in the small bowel. which is atypical for those who did, the cessation of the antibiotic itself may A A D o r CDAAD. Thus. S. uurer~smay be an infrequent have been the I-eason for thc diarrhea resolution. Othcr etioloey of AAD. but morc research is needed. studies havc found high stool counts oC C riii~ic~rr7,r and no otlier enteric pathogen when diarrhea was present. Clorrri~lilrnipei:/i.irr,yn.c The counts of C aihicur7.v decreascd after treatment u:ith The association between C: pcrfiingeiu and AAD has oral antifungal treatment with resolution of diarrhea. only bccn reported in a fcm small studies [79, 801. Samuel Recurrence of diarrhca corresponded with a rctitrn of ct al. [80] reported 25 cases of C. pafii~rgens-associated high counts of C all?iccin.r [70, 711. Possible mechanisms dial-rhea. of whom 72'X had been exposed to antibiotics of C. nihicorrs diarrhea may involve the impairment of (penicillins. ccphalosporins. a~iioxycillinlclavulanate.erysodiuni and water absorption due to this yeast or llic thromycin. etc.). The cases fit illto a similar risk profile ability of C albicrtn.s strains to p~.oducchigh levels of P- for patients with CDAAD, but were C di[fil,ile negative. lactamases [70, 721. Rendornized trials comparing tl-eat- The paticnts were older ( > 70 years old). generally fernale metlts with antifungals and placebo or antibiotic discon- ;tnd were close in proximity to other infected patients. tinuation may be required beforc the role of C: rtihicnr7.s However. o f t h c 200 patients witli infective diarrhea (not is fully resolved. all rcccived antibiotics), only 12.5'%,of thc cases were attributable to (1 pri:Jbirig~~ris [80]. These limited data do Klehsiellrr os,~'tocu pcrfi.iniger?.r as an etiology of not support the role of In rare occasions, KI~~h.siellir has been associated with AAD. but fut-ther sti~diesare needed to conclusively rule A A D [73-751. Benoit et al. [74] examined 20 paticnts out this organism. with A A D and 36 control patents by colonoscopy or of tlie ,Lli.sr.cll(rricol,.vE~'liologi
'

Review of AiZD

Dl? 1 3 1 5

1098: 16292 111:

295

agent. Investigation of viral etiologies of AAD may be rruitful, but are hampered by nosocomial outbreaks such as rotaviral infections frequently seen in pediatric patients [45, 471.

Metuholic Allerrrfions The effect of disturbing the normal flora may also result in the decrease in absorbable short chain fatty acids (SCFA) and the increase in nonabsorbable carbohydrates that may lead to osmotic diarrhea [5, 821. A decrease in SCFA has been observed in patients exposed to antibiotics who develop CDAAD and AAD [70, 82. 831. Clausen et al. [S2] studied patients with AAD and found significantly lower SCFA concentrations in the stool compared to nondiarrheal controls (22.1 and 59.5 nnnolil, p i 0.01, respectively). Broad-spectrutn antibiotics, which disrupt a greater range of normal flora, are associated with a significa~itdecrease in SCFA, whereas narrowspectrum antibiotics do not affect SCFA concentrations. SCFA have been shown to be a potent stimulator of water and electrolyte absorption, thus a decrease in SCFA may result in diarrhea caused by the lack of water absorption [84, 851. The disruption of the normal flora may also result in a decrease in the fermentation of largc carboliydrates (such as wheat starch). The increase in these large nonabsorbable carbohydrates has been shown to result in osmotic diarrhea [86]. Otlier Mechani.~msof A A D Also, antibiotics may affect the enteric nervous system which is involved in normal intestinal transit and physiology [87]. Percy and Christensen [88] reported that ampierythromycin and lincomycin were cillin, clindamyci~~, capable ofdepressing muscle tone and neuroeffector transmission to the intestinal muscularis mucosa. This finding may have been due to infection with toxigenic C ~lificile rather than a direct effect of the antibiotic. but assays for C cl;fFcile were not performed. Toxin A was later shown to increase myoelectric activity and be involved in capsaicinsensitive sensory afferent neurons and mast cells in the intestine [89]. Erythromycin has also been shown to have an effect on motility which varies by the region of the gastrointestinal tract [90]. Further investigation on the role of the enteric nervous system and AAD are needed.

Clinical Features AAD has a spectrum of severity including uncomplicated diarrhea, colitis and PMC. Unconiplicated diarrhea

296

Dig Dis 1998:16:292-307

has the highest incidence in both CDAAD and nonCDAAD (10-301100 patients), while colitis (which is usually CDAAD) is less frequent (5-101100 patients) and PMC (almost always CDAAD) is infrequent (0.1-11100 patients). AAD can be defined as diarrhea associated with recent antibiotic use (typically < 2 months) with no evidence of colitis or pseudomenibranes. Diarrhea has been defined differently in the literature but generally refers to a change in the normal stool frequency with at least 3 loose or watery stoolslday for several days [S, 32, 361. Stools may be foul-smelling or greenish, with or without mucus, low-grade fever and rare presence of blood [37]. The incubation time (defined as the time between antibiotic initiation and the onset of diarrhea) ralls into two groups: early onset, occurring during antibiotics treatment, and delayed onset, which may occur from 2 to 6 weeks after the antibiotics have been discontinued [4, 23. 381. In a study of patients on p-lactam antibiotics. the mean incubation period for AAD was 14.2 days (ranging from 2 to 55 days) [S]. The majority of the patients developed AAD while on the antibiotics (n = 13, 62O/;)), but 8 (38%) of patients did not develop AAD until after the antibiotics had been discontinued. The median duration of AAD was 4 days if diarrhea occurred while on the antibiotics and a median of 18 days if AAD occurred after antibiotics treatment [S]. Although AAD not associated with C. d18cile has not been associated with serious problems, complications of CDAAD have included protein-losing enteropathy and arthritis [38, 91-93]. If colitis develops, the diarrhea is usually more severe and associated with abdominal painlcramping, fever exceeding 40 "C, hypoalbuminemia and leukocytosis [37]. Colitis can be diagnosed by colonoscopy when inflammatory changes (erythema, friability or edema) are noted by biopsy but no pseudomembranes are present [38, 941. Histologic examination for C dlficile colitis may reveal 'summit lesions' or 'volcano lesions' where inflammatory cells and debris are ejected into the colonic lumen [37]. Sigmoidoscopy of patients with K. oxytocr~usually reveals diffuse or segmental left colitis [73]. At the extrei~leend or the spectrum of severity is PMC, which almost always is associated with C. d~lfficile.The symptoms of PMC include watery diarrhea (90-95%), abdoininal cramping (80-901%3,fever (80%). leukocytosis (801%,)and, rarely, voinitiiig [13. 951. Most patients with PMC become symptomatic within I week after antibiotic exposure, but symptoms may be delayed in as many as 40% of patients for 2-8 weeks after antibiotics have been discontinued [95]. Complications of PMC may include hypokalamia (37%). renal railu~z(27'%), or hypoprotei-

McFarland

Table2. Diag~iosisofantibiotic-associatcddiiirrheaa n d antibiotic-associaled diarrhea

(:~ l i t f ~ r i l i upon t o I-ulc out C rl[fficile. Enzyme immu~loassaytests for C dificiir toxins A and B have bcen shown to have

63-99'X, sensitivity and 75-100% specificity [36, 981. Although fe\a: local niicrobiology laboratories perform culMedical history History of recent antibiu~ics( < ? 111onlhs) tures for C. dificiir, up to 38'% of cases may be missed if cultu1-es are not done [7]. Exaniination of the stools Histot-y ol.reccnt hospit;lliration - .. for leukocytes may not be sutficiently specific to diagnosis Excludc other Medic;itions resulting in dial-rhe;i C riuficiic infections by itselC but may be clinically useful diarrhca c:iuses Chronic intestinal conditions for suspccted CDAAD and to separate etiologies for in(inflammatory bouel discasc. Clahn.5. S. u~~reirv) and non-inflammaflammatory (Srrii~io~rell~~, ischemic colitis. short bowel synclromc) Food intolerances tory diarrhea (C.jlciji.i~igms)[99]. The sensitivity of fecal - leultocytcs for CDAAD is low (28-35'%1) and may vary Exclude nonanribiotic S ~ ~ ( l k ~ ~S/:lli,qc'Nted PMC. Gasrroenterolosy DN. ealginni J N : Decrease in nosocomial Clalo13. 0h;)hc i \ L n t c r o l o x i c ; ~ u l ~ ~oi ri ytiliib90 C1i;~rles F. Pl~iilipsSF. Catnilleri M. Thola62 Bartlett J(i: (%rrrV/i8iiii iii!iiciir~: I-listory o i il, \;0110 c o ~ r , , ~ c~ y l o t ~ ~8 i ,~ rabbi1 ~ r ~ i ~ ~ l c ~ l i r ~ ~ fordr ~ l C M : Rapid gasll-ic emplyin: i n pnticnls ~.olcas an enteric p a t l r a g c ~and ~ ihecut.t.u#~i sl;lle I c i a 1 1 1 1 . Mayo Clin I'mc Inolb. lniccl Im8niiw 19'14:62:172-177. 1~J'iI:72:2Zi-3ZS. o f kno~vledgc;$bout t l ~ cor$;tnism. Clio lnicct 76 Yoko);~ln;i T. K o r ~ l oH . \'ohoI;t T. Tokuc Y, %tito D . S1linl:~Is \' S u p ~ l n ~ K r ;:~Colon*copy 91 Ct.on RQ. G o d o n PV: Rcisctive arlhrilir l o D i r 1994:18(~1~ppl4):?65-272. iilv rmnk hl~n,dy rtoolr .~\\oui;,ted ir!tll cancer Clr~~,ri,/i!otz di[ri~lsl>c(.. IJlru~lom~pa urology 198'):i)h:YXI-988. litis i.il~8\rrririiirri,iiriiii i*.r/roi,q,,ii< cnlcrotmiao f surgery i n solibiotic-iadocrd pseudomcmSteinel TS. Fontelen M C : Dinrrlics. dernop;isroc~iacddii~~.rl~uc;l J H o r p Infccc 1901: 18: branous cnlaocolilir. A m J S u g l99il;lbO: 219-2.;0. 535 i31J. rephy andcell signsl~ng:Lessons t i i > ~ n i n i c l o b ~ i i l lox~ns.Tr.~or All? Clin Clim;iloi A u o c 1997.108: 81 Kitlo S. Ehiii;~K. (lriw;, ,\. U;~p;marna H . U;t96 1 ktyrvnrd US. Wcnsel R11. Kihsey 1:' Kclaprine 149 164. k;$g;iwa H: A n t ~ h ~ o l ~ r . i ~ s r o c i lhct~~oirb;igic iii~c~i ~ ' l , , . ~ ~ r i ~,i8(iicil I ? nraloull-iill-l-llrir. incluJb~l$rnolecul:~l-iliagnostic methhil~ry,Clin Infect Dis I9~J5:21:XSI-886. lion. G ; ~ ~ l n ~ t ~ ~ i t c r oI')Xu:97:62? lap! $10. uda i!o