- Email: [email protected]
Identification of the cause of a brain abscess by direct 16S ribosomal DNA sequencing
Case Reports 16 ‘l’ajiki MH. Salomao R. Association of plasma levels of tumor necrosis factor alpha with severity of disease an6 mortality among patients with leptospirosis. C’lirr Ir~jbr‘t Dis 1996: 23: 1 177-11 7X. 17 Friedland IS. \Varrell DA. The Jarisch-Herxheimer reaction in leptospiroais: possible pathogcncsis and review. Kw Ir!(iw Dis 1 YY 1: 13: 207-l IO. 18 \h’akahayashi Y. Kamijon Y, Soma K. Ohwada ‘I’. Kernoval of circulating cytokines by continuous haemoliltration in patients with systemic infkrmmatory response or multiple organ dysfunction syndrome. I3r- 1 Surq 1YYh: 83: 3Y 3-394. IY Kourdais A. Lonjon 13. L’crgcs-Pascal R. Fournier A. Ah 1.0 W. Respiratory complications of leptospirosis. Apropos of 6 cases. 3 of which show hemodynamic studies. !\/lerl ‘I’rq~ (,Wnrs) lYX8: 48: 14Y-1 60. 20 Davenport A. Bramley I’N. Wyatt JI. Morbidity and mortality due to cerchral edema complicating the treatment of severe leptospiral infection. ,4m 1 Kiifrwg I1i.s 1 YYO: 16: 1 hOk1 65.
21 Ogura
E1. Ciofli WG. Offner 1’1 it trl. Effect of inhaled pulmonary function after sepsis in a swine model. 116:
nitric oxide on Surq!~prq 1 YY4:
313-321.
22 Bloomfield
23 24 25 26
GL, Holloway S. Ridings PC’ vt rd. Pretreatment with inhaled nitric oxide inhibits ncutrophil migration and oxidatiw activity resulting in attenuated sepsis-induced acute lung injury. Crit Cwe ,l/lrrf 199 7: 25: 5X4-59 3. Krafft I’. Fridrich 1’. Fitzgerald RI). Koc I). Steltxer H Elktiveness ol nitric oxide inhalation in septic ARDS. Cl~~,st 1996: 109: 48639 3. Turner JS, Willcox PA. Respiratory failure in leptospirosis. (! / !\;Icv/ 1YXY: 269: X41-X47. Ikrcndsen NH. Rommes JH. Hykcmabs PI rrl. Adult respiratory failure and leptospirosis. Ann lfltcrrl .Llcd 1 Y X4: 101 : 402. Zaltzman M. Knllenhach JM. Goss Gl) rat (I!. Adult respirtttory distress syndrome in leptospira canicola infection. Xr ,t21ctf] 19X1: 2X3: ilY-520.
Identification of the Cause of a Brain Abscess by Direct 16s Ribosomal DNA Sequencing J. M. J. Logan*, G. V. Orange and A. F. Maggst
We report the case of a young man who apparently suffered successive episodes of meningitis and cerebral abscess over a l-month period, both of which were diagnosed by two different molecular approaches: PCR for ~Ncisscrin meniryitidis IS I IO6 from CSF and 16s rRNA gene sequencing on a specimen of brain pus. In each case, cultures were negative due to prior antibiotic therapy.
Introduction Whilst still only relevant to a minority of specimens. molecular techniques are becoming increasingly used in medical microbiology. One of their main strengths is the ability to provide answers shortly after antibiotics have been used where traditional cultural techniques are negative. The polymerase chain reaction (PCR) has the ability to detect a pathogen in a clinical sample by targeting genes that are ‘specific’ to that organism, such as the insertion sequence (ISI IO(i) that was thought to be found only on the chromosome of Neissuia rurwiryitiriis. A contrasting approach is to use a universally conserved target for PCR. such as the IhS rRNA gene. and then to use DNA sequencing of the product to identify the pathogen involved. \Ve describe a patient in which both techniques were used to diagnose a central nervous system infection.
Case Report A 3 T-year-old previously well man was admitted to hospital with headache, fever. neck stiffness and a j-day history of flulike illness. Six days prior to admission. he had undergone * Address all correspondence to: Julie IM. J. Logan. Molecular Biology (Init. Virus Refcrencc IIivision. Central Public Health Laboratory. 61 C’olindale Avenue. London NWY 5IIT. 1l.K. j- Current address: Department of Microbiology and Immunology. IAcester llniversity Medical School, Medical Sciences Ruilding. Leiccster 1X1 YHN. T1.K. Accepted for publication 16 September 1 Y Y 8.
extraction of a molar tooth without antibiotic prophylaxis. There was a history of childhood epilepsy and a cardiac murmur but echocardiography 10 years earlier had not shown any significant abnormality. On examination he was pyrexial and slightly cyanosed (0, saturation Xh’% on air). he had neck stiffness but Kernig’s sign was negative and there were no focal neurological signs. Three small petechiae were noted on the buttocks. The General Practitioner had suspected meningitis and administered I .2 g benzylpenicillin II’ prior to admission and the patient received 1 g cefotaxime empirically on arrival at hospital. A computerized tomography (CT) scan was not performed. but blood cultures and cerebrospinal fluid (CSP) were obtained for microbiological examination. The CSF white cell count was raised at 1.24 x IO”/1 (6 5% polymorphs) but no micro-organisms were seen on microscopy. Blood cultures and CSF culture subsequently yielded no growth and the CSF supernatant was submitted to the Scottish Meningococcal Reference Laboratory for PCR examination. Species-specific PCR for meningococci based on the IS1106 gent target’ was positive confirming the suspected diagnosis of N. rtmir~yititlis meningitis. The patient improved clinically on cefotaxirne. On further examination a soft systolic murmur was noted at the left sternal edge and echocardiography showed tricuspid regurgitation and right ventricular hypcrtrophy but no valvular lesions or evidence of intracardiac shunt. The patient was discharged home 15 days after admission having received 2 weeks of IV cefotaxime therapy and a single oral dose of ciprolloxacin to eradicate presumed nasal carriage of N. rmwiryitirlis. Nine days later the patient was readmitted with a 24-hour
46
I 0.54’ sequence divergence
R Asii
history of severe frontal headache having suffered two rightsided focal tits that day. On examination he was drowsy and pyrexial with no ncclc stiffness and Kernig’s sign was negative. A cerebral abscess \~as suspected and the patient was transferred to the nearest neurosurgical unit. CT scanning showed the presence of a possible left frontal lobe abscess which was biopsicd under CT guidance and the patient commenced on IV cefuroxime and metronidazole empirically. Tissue was obtained that showed a non-specific cerebritis histologically and yielded no growth on microbiological culture. The reference laboratory found the tissue to be IS 17 06 PCR positive but IgG enzymclinked immunosorbent assay (LLISX) positive and IgM ELlSA negative against meningococcal outer membrane protein. Blood was also sent to the refercncc laboratory. and lbund negative on testing for both IS1106 PCR and latex agglutination for meningococcal antigen. A repeat CT scan 11 days later showed the presence ofa definite cerebral abscess in the right parafalcinc region which was then drained. The pus obtained yielded no growth on culture and the reference laboratory found it to bc ISllOh PCR negative. During this time, DNA was extracted from the pus and subjected to PCR amplilication using universal bacterial 1 hS rRNA primers’ and the product was sequenced on an Applied Biosystems model 3T3A automated DNA sequencer. The sequence obtained was compared with other t f2S rRNX sequences in the GcnEank/EMRI, databases and a dcndrogram constructed based on sequence similarity. The unrooted tree from the 448 base pair overlap with primers POmodil’C 3mod’ is shown in l:igurc 1. From the dcndrogram it is clear that the sequence obtained directly from the brain pus sample is most closely related to the group of four established oral I’[rsohar,tc,rirlr~~ species. Antibiotic therapy MGIS changed to ccfotaxime and mctronidazole following drainage of the abscess and the patient was discharged home after h weeks of treatment. The time course of this case and laboratory results arc summarized in Table I.
//
‘El ulcerans l? uarium
He later underwent further cardiac investigations including cardiac catheterization which showed that he had an inter-atria1 septal aneurysm and severe pulmonary arterial hypertension of uncertain aetiology. There was no atrial septal defect but the presence of a patent foramen ovale was not ruled out. In an 1 X-month followup period he has had no further episodes of sepsis. and continued to require anticonvulsant therapy together with high dose vasodilator therapy for presumed primary pulmonary hypertension.
Discussion This is ;m interesting case in which all attempts at culture proved negative and molecular tools were used to provide :I diagnosis. Direct sequencing of the brain pus yielded a DNA sequence compatible with the presence of a I:tlsobirc,l(‘rilrrrl spp. Although a false-positive 1 fd rRNA gene I’CK cannot be excluded, it is unlikely in this instance as fusobacteria have never been previously gromrn or detected by VCR in this laboratory and this organism is no1 a common environmental cow taminant. This agent had to have been present alone or, if other bacteria were present, their numbers were extremely low in relation to the I~usclh~~trritrrt~. otherwise an unreadable or ambiguous sequence would have been obtained. (Xven the history and the anatomical position, it is likely that this cerebral abscess was dental in origin. The sequence amplilied was closely related to the oral group of four established ~~‘lrsohr~c~rc~ri~rrrl species. which exhibit high levels of 1hS rRNA sequence identity to each other ranging from Y7.3 to YY. 5%. whereas their identity to other fusobacterial species is lower at Y 1.X to Y4.h’%. As sequence identity is so high it is diflicult accurately to speciatc this organism within the oral group. although overall scqucncc similarity would suggest the abscess was caused by II r~wlcatl~t~. Fusobactcria are important patho-
I,. Smart. Scotlish hlcningococcal Kcl‘crcnw li)r the tests l’or \‘. rtwrtirt~gitirfi.\ and Itelpl’~~l discussions. Also thanks lo I? I-. C’nrlcr. lIepat-ttiicwt of Zldical ~licrol~iology. lltii\-rrsity of Ahcrdecti for asxistaticc with xqueticitig. ‘l’his work henelileti l’rom the use ()I’ the SlX]NlX ktcilit) at C’entt-al Laboralory of the Kescat-ch ~‘ouncils. I)xeshut-!, We wish
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the dc~~~clopmetil of the i,et-ebral abscess is not clear. Aletiitigitis is cct-lainly ii rat-c primal-!. c\~eitl Icacliti, (1 to hraiti abscess. as the pia tnatcr forms it substantial hat-t-ier lo progression ol itikcdiott lo 1he ccrchrutn. but il is possible lhktl the tiietiitigococc;iI ittldioti may ha\rc created the initial damage allo\vitig the l’usohactct-ial ahsccss to progress. Howcvcr. Iherc MYIS no c\~idcttcc ol’ttwtlitlgococcal l)‘u,-\ king prcwnl once the abscess had hecome cstahlishcd anti it is intcresling to note that twititigococcal lg.21 \z’i~s trticlctcctahlc b!r I3.ISA some ~4~eelts postittfcc(ion. ,\ ktr~hvr csplanalion might he ct-ass-h\lbridixation bcl12~ecii the IY’K primers aitncd at the t~ictiiti~ococcaI itiscrtioti seqtrciiw and a scquctic~e iti I~tr.so/~cfc~Ic~r.it~~tt. The IS 7 706 perliti-tiicd iis originall!, dchct-ihd Iiits kcti slio\zn to hii\,c prohlems \z,illi ii sinall ~it~t~il~eroffltlse-positi\,cs”ntid the ititroduc~ioti or ;I prohittg stc’p in a IY’K-I:I,IS.\ l’ot-ma(” and improved desigti 01‘ pritiwrs atid probe’” Iiii\‘c’ not cotiipletel~~ elitiiiti~tted this prohktii. tresul(iti g iti Ilie suhsequetil u?thdrawal 0fIhis assay.” ‘I’llc most probahlc csplatlatiotl for the tncningococcal IY’K result is that iI \z’;is ii liilsc-positi\,c. ‘l’lic LISA’ ol‘~rtilihiotics priot- io samples king taken IOr tiiicrobiological cult~rrr is common. In sottic’ cast‘s Ihis is consistent \I,ith good ic~tttetnporat-1 clinical pr;tctice. bul it does tiicwti Ihat the diagnosis is lcxs lildl) to be achiclwi through cultural mrlhotis. ~lolcc~dtr hiologic~rl techtiiqitcs cat1 tlicti he extrctiiely hclpl’ttl. ol’lcti pi-o\riditig the onI!, tnt’atis 01‘establishitig it dclitiiti\ c diagnosis. tto\vever. this cxsc’ dctnonstratcs both the power and pitkills 0I’ (licse tnclliods. As mot-c and mow ~iiethods hccotnc a\~ail;ible. their skilled ititerprt~ta~ioti alongside Ilic clitiical pi-cscti~atiott \li,ill lwcoinc iticreasiti~ly itiiporlatit. j~rst as \z’ith lahora~ory rcst~lIs oh~aiticd hg mot-c traditional tncittis.
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21 Ogura
E1. Ciofli WG. Offner 1’1 it trl. Effect of inhaled pulmonary function after sepsis in a swine model. 116:
nitric oxide on Surq!~prq 1 YY4:
313-321.
22 Bloomfield
23 24 25 26
GL, Holloway S. Ridings PC’ vt rd. Pretreatment with inhaled nitric oxide inhibits ncutrophil migration and oxidatiw activity resulting in attenuated sepsis-induced acute lung injury. Crit Cwe ,l/lrrf 199 7: 25: 5X4-59 3. Krafft I’. Fridrich 1’. Fitzgerald RI). Koc I). Steltxer H Elktiveness ol nitric oxide inhalation in septic ARDS. Cl~~,st 1996: 109: 48639 3. Turner JS, Willcox PA. Respiratory failure in leptospirosis. (! / !\;Icv/ 1YXY: 269: X41-X47. Ikrcndsen NH. Rommes JH. Hykcmabs PI rrl. Adult respiratory failure and leptospirosis. Ann lfltcrrl .Llcd 1 Y X4: 101 : 402. Zaltzman M. Knllenhach JM. Goss Gl) rat (I!. Adult respirtttory distress syndrome in leptospira canicola infection. Xr ,t21ctf] 19X1: 2X3: ilY-520.
Identification of the Cause of a Brain Abscess by Direct 16s Ribosomal DNA Sequencing J. M. J. Logan*, G. V. Orange and A. F. Maggst
We report the case of a young man who apparently suffered successive episodes of meningitis and cerebral abscess over a l-month period, both of which were diagnosed by two different molecular approaches: PCR for ~Ncisscrin meniryitidis IS I IO6 from CSF and 16s rRNA gene sequencing on a specimen of brain pus. In each case, cultures were negative due to prior antibiotic therapy.
Introduction Whilst still only relevant to a minority of specimens. molecular techniques are becoming increasingly used in medical microbiology. One of their main strengths is the ability to provide answers shortly after antibiotics have been used where traditional cultural techniques are negative. The polymerase chain reaction (PCR) has the ability to detect a pathogen in a clinical sample by targeting genes that are ‘specific’ to that organism, such as the insertion sequence (ISI IO(i) that was thought to be found only on the chromosome of Neissuia rurwiryitiriis. A contrasting approach is to use a universally conserved target for PCR. such as the IhS rRNA gene. and then to use DNA sequencing of the product to identify the pathogen involved. \Ve describe a patient in which both techniques were used to diagnose a central nervous system infection.
Case Report A 3 T-year-old previously well man was admitted to hospital with headache, fever. neck stiffness and a j-day history of flulike illness. Six days prior to admission. he had undergone * Address all correspondence to: Julie IM. J. Logan. Molecular Biology (Init. Virus Refcrencc IIivision. Central Public Health Laboratory. 61 C’olindale Avenue. London NWY 5IIT. 1l.K. j- Current address: Department of Microbiology and Immunology. IAcester llniversity Medical School, Medical Sciences Ruilding. Leiccster 1X1 YHN. T1.K. Accepted for publication 16 September 1 Y Y 8.
extraction of a molar tooth without antibiotic prophylaxis. There was a history of childhood epilepsy and a cardiac murmur but echocardiography 10 years earlier had not shown any significant abnormality. On examination he was pyrexial and slightly cyanosed (0, saturation Xh’% on air). he had neck stiffness but Kernig’s sign was negative and there were no focal neurological signs. Three small petechiae were noted on the buttocks. The General Practitioner had suspected meningitis and administered I .2 g benzylpenicillin II’ prior to admission and the patient received 1 g cefotaxime empirically on arrival at hospital. A computerized tomography (CT) scan was not performed. but blood cultures and cerebrospinal fluid (CSP) were obtained for microbiological examination. The CSF white cell count was raised at 1.24 x IO”/1 (6 5% polymorphs) but no micro-organisms were seen on microscopy. Blood cultures and CSF culture subsequently yielded no growth and the CSF supernatant was submitted to the Scottish Meningococcal Reference Laboratory for PCR examination. Species-specific PCR for meningococci based on the IS1106 gent target’ was positive confirming the suspected diagnosis of N. rtmir~yititlis meningitis. The patient improved clinically on cefotaxirne. On further examination a soft systolic murmur was noted at the left sternal edge and echocardiography showed tricuspid regurgitation and right ventricular hypcrtrophy but no valvular lesions or evidence of intracardiac shunt. The patient was discharged home 15 days after admission having received 2 weeks of IV cefotaxime therapy and a single oral dose of ciprolloxacin to eradicate presumed nasal carriage of N. rmwiryitirlis. Nine days later the patient was readmitted with a 24-hour
46
I 0.54’ sequence divergence
R Asii
history of severe frontal headache having suffered two rightsided focal tits that day. On examination he was drowsy and pyrexial with no ncclc stiffness and Kernig’s sign was negative. A cerebral abscess \~as suspected and the patient was transferred to the nearest neurosurgical unit. CT scanning showed the presence of a possible left frontal lobe abscess which was biopsicd under CT guidance and the patient commenced on IV cefuroxime and metronidazole empirically. Tissue was obtained that showed a non-specific cerebritis histologically and yielded no growth on microbiological culture. The reference laboratory found the tissue to be IS 17 06 PCR positive but IgG enzymclinked immunosorbent assay (LLISX) positive and IgM ELlSA negative against meningococcal outer membrane protein. Blood was also sent to the refercncc laboratory. and lbund negative on testing for both IS1106 PCR and latex agglutination for meningococcal antigen. A repeat CT scan 11 days later showed the presence ofa definite cerebral abscess in the right parafalcinc region which was then drained. The pus obtained yielded no growth on culture and the reference laboratory found it to bc ISllOh PCR negative. During this time, DNA was extracted from the pus and subjected to PCR amplilication using universal bacterial 1 hS rRNA primers’ and the product was sequenced on an Applied Biosystems model 3T3A automated DNA sequencer. The sequence obtained was compared with other t f2S rRNX sequences in the GcnEank/EMRI, databases and a dcndrogram constructed based on sequence similarity. The unrooted tree from the 448 base pair overlap with primers POmodil’C 3mod’ is shown in l:igurc 1. From the dcndrogram it is clear that the sequence obtained directly from the brain pus sample is most closely related to the group of four established oral I’[rsohar,tc,rirlr~~ species. Antibiotic therapy MGIS changed to ccfotaxime and mctronidazole following drainage of the abscess and the patient was discharged home after h weeks of treatment. The time course of this case and laboratory results arc summarized in Table I.
//
‘El ulcerans l? uarium
He later underwent further cardiac investigations including cardiac catheterization which showed that he had an inter-atria1 septal aneurysm and severe pulmonary arterial hypertension of uncertain aetiology. There was no atrial septal defect but the presence of a patent foramen ovale was not ruled out. In an 1 X-month followup period he has had no further episodes of sepsis. and continued to require anticonvulsant therapy together with high dose vasodilator therapy for presumed primary pulmonary hypertension.
Discussion This is ;m interesting case in which all attempts at culture proved negative and molecular tools were used to provide :I diagnosis. Direct sequencing of the brain pus yielded a DNA sequence compatible with the presence of a I:tlsobirc,l(‘rilrrrl spp. Although a false-positive 1 fd rRNA gene I’CK cannot be excluded, it is unlikely in this instance as fusobacteria have never been previously gromrn or detected by VCR in this laboratory and this organism is no1 a common environmental cow taminant. This agent had to have been present alone or, if other bacteria were present, their numbers were extremely low in relation to the I~usclh~~trritrrt~. otherwise an unreadable or ambiguous sequence would have been obtained. (Xven the history and the anatomical position, it is likely that this cerebral abscess was dental in origin. The sequence amplilied was closely related to the oral group of four established ~~‘lrsohr~c~rc~ri~rrrl species. which exhibit high levels of 1hS rRNA sequence identity to each other ranging from Y7.3 to YY. 5%. whereas their identity to other fusobacterial species is lower at Y 1.X to Y4.h’%. As sequence identity is so high it is diflicult accurately to speciatc this organism within the oral group. although overall scqucncc similarity would suggest the abscess was caused by II r~wlcatl~t~. Fusobactcria are important patho-
I,. Smart. Scotlish hlcningococcal Kcl‘crcnw li)r the tests l’or \‘. rtwrtirt~gitirfi.\ and Itelpl’~~l discussions. Also thanks lo I? I-. C’nrlcr. lIepat-ttiicwt of Zldical ~licrol~iology. lltii\-rrsity of Ahcrdecti for asxistaticc with xqueticitig. ‘l’his work henelileti l’rom the use ()I’ the SlX]NlX ktcilit) at C’entt-al Laboralory of the Kescat-ch ~‘ouncils. I)xeshut-!, We wish
Laboratory
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trc‘Ic\‘ilIlcc’
01’ the
\‘.
rllc’rtirt~/ilirli.s
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the dc~~~clopmetil of the i,et-ebral abscess is not clear. Aletiitigitis is cct-lainly ii rat-c primal-!. c\~eitl Icacliti, (1 to hraiti abscess. as the pia tnatcr forms it substantial hat-t-ier lo progression ol itikcdiott lo 1he ccrchrutn. but il is possible lhktl the tiietiitigococc;iI ittldioti may ha\rc created the initial damage allo\vitig the l’usohactct-ial ahsccss to progress. Howcvcr. Iherc MYIS no c\~idcttcc ol’ttwtlitlgococcal l)‘u,-\ king prcwnl once the abscess had hecome cstahlishcd anti it is intcresling to note that twititigococcal lg.21 \z’i~s trticlctcctahlc b!r I3.ISA some ~4~eelts postittfcc(ion. ,\ ktr~hvr csplanalion might he ct-ass-h\lbridixation bcl12~ecii the IY’K primers aitncd at the t~ictiiti~ococcaI itiscrtioti seqtrciiw and a scquctic~e iti I~tr.so/~cfc~Ic~r.it~~tt. The IS 7 706 perliti-tiicd iis originall!, dchct-ihd Iiits kcti slio\zn to hii\,c prohlems \z,illi ii sinall ~it~t~il~eroffltlse-positi\,cs”ntid the ititroduc~ioti or ;I prohittg stc’p in a IY’K-I:I,IS.\ l’ot-ma(” and improved desigti 01‘ pritiwrs atid probe’” Iiii\‘c’ not cotiipletel~~ elitiiiti~tted this prohktii. tresul(iti g iti Ilie suhsequetil u?thdrawal 0fIhis assay.” ‘I’llc most probahlc csplatlatiotl for the tncningococcal IY’K result is that iI \z’;is ii liilsc-positi\,c. ‘l’lic LISA’ ol‘~rtilihiotics priot- io samples king taken IOr tiiicrobiological cult~rrr is common. In sottic’ cast‘s Ihis is consistent \I,ith good ic~tttetnporat-1 clinical pr;tctice. bul it does tiicwti Ihat the diagnosis is lcxs lildl) to be achiclwi through cultural mrlhotis. ~lolcc~dtr hiologic~rl techtiiqitcs cat1 tlicti he extrctiiely hclpl’ttl. ol’lcti pi-o\riditig the onI!, tnt’atis 01‘establishitig it dclitiiti\ c diagnosis. tto\vever. this cxsc’ dctnonstratcs both the power and pitkills 0I’ (licse tnclliods. As mot-c and mow ~iiethods hccotnc a\~ail;ible. their skilled ititerprt~ta~ioti alongside Ilic clitiical pi-cscti~atiott \li,ill lwcoinc iticreasiti~ly itiiporlatit. j~rst as \z’ith lahora~ory rcst~lIs oh~aiticd hg mot-c traditional tncittis.
I.abot-a1or~~.
I
1
;
7
s
9
IO
‘l’his worl\ tiealtllcat-c and llcallh
148~1s partly l’undcd Kcswrch ~‘otiitnittec Ikpartttien~.
hy
a grant
01‘ Ihe
Scoltish
l’rotn
the
Acute
Oflice
Home
11
to thank