Case Report/Clinical Techniques

Infection in a Complex Network of Apical Ramifications as

the Cause of Persistent Apical Periodontitis: A Case Report
Michael Arnold, Dipl Stom,* Domenico Ricucci, MD, DDS,† and Jos
e F. Siqueira, Jr, DDS, MSc, PhD‡

Abstract
Introduction: This article reports a case of persistent
apical periodontitis lesion in a mesiobuccal root of
a maxillary molar subjected to single-visit endodontic
P rimary and posttreatment apical periodontitis lesions are primarily caused by
microbial infection of the root canal system. The successful outcome of endodontic
treatment depends on thorough disinfection of the root canal system (1). Although it
treatment. Methods: The treatment protocol followed has been suspected that persistent infection can be related to resistant and more robust
endodontic standards including using nickel-titanium microbial species present in the canal (2, 3), there is ample evidence that the main (or
instruments with working length ending 0.5-mm short most common) reason for bacterial persistence after treatment is the fact that infection
of the apex, establishment and maintenance of apical can spread to areas of the root canal system that remain unaffected by instruments and
foramen patency, irrigation with 5% NaOCl, smear layer antimicrobial substances used for irrigation or medication (4, 5). These include not
removal, a final rinse with and ultrasonic agitation of only untouched walls of the main canal (6) but also areas distant from the main canal,
chlorhexidine, and filling by the vertical compaction such as lateral canals (7, 8), apical ramifications (9, 10), isthmuses (6, 11), and
technique. Even so, the lesion in the mesiobuccal root dentinal tubules (8, 12). The present case report is about a persistent posttreatment
became larger in size after follow-up examination at 1 apical periodontitis lesion caused by infection established in a complex apical root
year 6 months, and periradicular surgery was performed. canal anatomy.
Radiographic control after 11 months showed that peri-
radicular healing was almost complete. The root apex Case Report
and the lesion were analyzed histologically and histo- A 51-year-old male patient was referred to an endodontic specialist by his general
bacteriologically. Results: The lesion was diagnosed dentist who had initiated root canal treatment of tooth #14. The patient presented to his
as a ‘‘pocket cyst,’’ and no bacteria were noted extrara- dentist for a routine checkup, and a review of his medical history was noncontributory.
dicularly. The cause of continued disease was a heavy The patient reported that more than 15 years earlier a prosthetic restoration was
bacterial biofilm infection located in an intricate performed in his upper left jaw, consisting of a bridge to replace teeth #11 and #13.
network of apical ramifications. Bacteria were also A recurrent deep caries lesion was diagnosed in tooth #14, and the patient declared
observed on the walls of one of the mesiobuccal canals no symptoms. A radiograph showed large periradicular radiolucencies on the
packed between the obturation material and the root mesiobuccal (MB) and palatal roots as well as a minor radiolucency on the distobuccal
canal wall. Conclusions: This case report reinforces root of tooth #14; the root canals appeared consistently narrowed (Fig. 1A). No
the need for treating the infected root canal as a complex radiographic signs of periodontal disease were observed. The diagnosis of pulp
system that possesses anatomic intricacies in which necrosis was made, and root canal treatment was indicated. An access cavity was
bacteria can spread and remain unaffected by treatment prepared through the existing restoration. The general dentist was not able to locate
procedures. (J Endod 2013;39:1179–1184) the orifices and negotiate the canals, so the patient was referred to an endodontist.
At the examination performed by the endodontist, the buccal mucosa did not show
Key Words any pathological changes. On the palatal side, the gingival margins were heavily
Apical delta, endodontic treatment, posttreatment inflamed, and bleeding occurred on probing. A carious lesion was detected on the
apical periodontitis, root canal infection, treatment palatal side, apical to the restoration margin, and the tip of a probe could penetrate
outcome the pulp chamber. The periodontal probing depth both mesially and distally was
4 mm. The tooth was not tender to percussion (vertical and lateral) or palpation
(buccal and palatal). On the bases of the existing diagnostic radiograph and clinical
From the *Private Practice, Dresden, Germany; †Private examination, the diagnosis of pulp necrosis with apical periodontitis lesions was
Practice, Cetraro, Italy; and ‡Department of Endodontics, confirmed for tooth #14, and root canal treatment was scheduled.
Faculty of Dentistry, Est
acio de S
a University, Rio de Janeiro, One week later, the bridge was sectioned, and the crown on tooth #14 was
Rio de Janeiro, Brazil. removed. After rubber dam isolation, carious tissue was excavated with low-speed
Address requests for reprints to Dr Domenico Ricucci,
Piazza Calvario, 7, 87022 Cetraro (CS), Italy. E-mail address:
round burs under water spray. The crown was then restored with composite
dricucci@libero.it (Tetric EvoFlow; Ivoclar Vivadent, Ellwangen, Germany) after acid etching and bonding
0099-2399/$ - see front matter application (Optibond FL; Kerr, Ratstatt, Germany). With the aid of an operating
Copyright ª 2013 American Association of Endodontists. microscope, the calcified tissue in the pulp chamber was removed, and 4 root canal
http://dx.doi.org/10.1016/j.joen.2013.04.036 orifices (2 in the MB root [MB1 and MB2]) were evident. The orifices appeared
obstructed by calcified tissue (Fig. 1B–D). The root canals were located by cautiously
removing the calcified tissue with ultrasonic diamond tips. After preparing the
orifices of MB1 and MB2, an isthmus connecting the 2 canals was evident (Fig. 1C).
The MB portion of this isthmus, which appeared patent, was opened with a prebent
#25 ultrasonic file (Irri K-Files; VDW, M€unchen, Germany) (Fig. 1D). Under

JOE — Volume 39, Number 9, September 2013 Apical Delta Infection and Treatment Failure 1179
Case Report/Clinical Techniques

Figure 1. (A) A radiograph of tooth #14 taken during a routine checkup by a general dentist showing periradicular radiolucencies. (B–D) A sequence of photo-
graphs showing the negotiation of MB1 and MB2 and the isthmus connecting the 2 canals as performed by an endodontist. (E) Gutta-percha cone selection and (F)
postobturation radiograph. (G) A 6-month follow-up radiograph. The radiolucencies on the distobuccal and palatal roots had considerably decreased whereas that
on the mesial root remained the same size. (H) An 18-month follow-up radiograph. The lesion on the MB root became larger. Apical surgery was scheduled. (I–K)
Cone-beam computed tomographic scans showing the extent of the lesion and its relationship with the maxillary sinus floor. (L and M) After elevating a mucoper-
iosteal flap, the cortical bone covering the pathologic tissue was carefully mobilized to create an access to the MB periradicular area. (N) The resected root end, (O)
the prepared root-end cavity, and (P) filling with MTA. (Q) A follow-up radiograph taken 11 months after surgery.

1180 Arnold et al. JOE — Volume 39, Number 9, September 2013

 Case Report/Clinical Techniques

Figure 2. (A) A mesial view of the apical biopsy including the MB root tip and the surrounding pathologic tissue in their original relationship. (B) A view of the
resected surface. A cavity can be observed in the soft tissue. (C) A section taken on a buccolingual plane encompassing the apical portion of the MB canal, a large
ramification, and the very apical portion of MB2 (arrow). The overview reveals that the lesion is a ‘‘pocket cyst’’ with its lumen in direct continuity with the root
canal space through the wide apical ramification (hematoxylin-eosin, original magnification 8). (D) A detailed view of the area from the cyst wall demarcated by
the rectangle in C. Stratified squamous epithelium with an arcading structure can be seen. The subepithelial connective tissue is infiltrated by inflammatory
cells (original magnification 50). (E and F) Progressive magnifications of the basal layer of the epithelial wall in D. The epithelium is infiltrated mostly by
polymorphonuclear leukocytes (arrowheads in F) (original magnification 400 and 1,000). (G) A section taken at a short distance from that shown in C (Taylor
modified Brown and Brenn, original magnification 8). (H) A detailed view of the root tip. MB1 bifurcates into 2 apical ramifications filled by a biofilm. The exit of
a wide ramification can be seen on the left profile as well as the entrance of a large ramification more coronally (original magnification 25). The area indicated by
the arrow is magnified in Figure 3E. (I) A detailed view of the 2 ramifications in H (original magnification 100).

microscopic observation, the isthmus seemed to end at the middle third The buccal root canals were instrumented apically up to #35.04
of the MB root canal system. and the palatal canal up to #45.04 using ProFile and FlexMaster rotary
The working length (WL) was established 0.5 mm short of the instruments in a step-down approach. During instrumentation, the
apical foramen using the electronic apex locator from the VDW Gold inte- canals were thoroughly irrigated with 5% NaOCl. Finally, all canals
grated system. The root canals were prepared with rotary nickel-titanium were irrigated with 10% citric acid to remove the smear layer followed
files (ProFile [Maillefer, Ballaigues, Switzerland] in combination with by a final rinse with 2% chlorhexidine. These solutions were activated
GTX [Dentsply Tulsa Dental Specialties, Tulsa, OK] and FlexMaster with ultrasonics for about 20 seconds each. The root canals were
[VDW]). A #08 K-file (Maillefer) was used to ensure patency of the apical dried with sterile paper points and filled with gutta-percha and
foramen by taking it 1 mm beyond the WL. The fourth canal (MB2) was 2Seal (VDW) using Schilder’s vertical compaction technique
found to have an independent course and foramen. (Fig. 1E and F). Subsequently, the tooth was restored with a DTLight

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Case Report/Clinical Techniques
SL post (VDW) and composite (Tetric EvoFlow). Finally, a temporary procedures. With the microtome set at 4–5 mm, longitudinal serial
crown was placed. sections of the apex with the surrounding pathologic tissue were taken
At the first follow-up visit 6 months later, the tooth was asymptom- on a buccolingual plane until the specimen was exhausted. Particular
atic. Percussion and palpation tests yielded normal responses. A new care was taken to obtain sections encompassing the foramen(ina) in
permanent crown was inserted by the general dentist. The radiograph conjunction with the pathologic periradicular tissue. Approximately
showed that the radiolucencies on the palatal and distobuccal roots 500 sections were cut in total for the apical biopsy. Sections of the
had consistently reduced, whereas the lesion on the buccal root had bone fragments were also cut on a buccolingual plane to observe the
remained the same size, even though the corticated margin was no contact area between the apical periodontitis lesion and the cortical
longer visible (Fig. 1G). buccal bone. Sections were stained with a modified Brown and Brenn
The patient returned after 1 year 6 months because 1 week technique for staining bacteria (13, 14). Selected slides were
previously he had noted buccal swelling and an active sinus tract in stained with hematoxylin-eosin. Slides were examined under the light
this region. At examination, no pathological signs were present. The microscope.
sinus tract was no longer present, and there was no tenderness to
palpation and percussion. Periodontal probing did not reveal pockets. Histopathologic and Histobacteriologic Observations
A periapical radiograph showed that although the lesions on the palatal
Microscopically, sections of the MB root tip revealed the presence
and distobuccal roots had apparently healed, the radiolucency on the
of a complex root canal system in the apical third, with an apical delta
MB root had become larger (Fig. 1H). Cone-beam computed
characterized by an intricate network of ramifications (Figs. 2C and G–I
tomographic imaging showed a large radiolucency associated mostly
and 3F). Numerous foramina could be observed in the serial sections
with the MB root apex, with corticated margins (Fig. 1I–K) and
ending at the geometrical top and on the palatal aspect of the root
elevation of the maxillary sinus floor (Fig. 1I). The case was regarded
tip. The ramification ending on the palatal aspect exhibited a large diam-
as root canal treatment failure, and apical surgery was scheduled for
eter and ended in a cavity surrounded by epithelium (Fig. 2C, G, and H).
1 week later.
The lesion had the characteristics of a ‘‘pocket cyst’’ with an epithelial
Oral disinfection was performed before treatment by rinsing with
lining adhered to the apical structure apically and palatally to form an
0.2% chlorhexidine (Chlorhexamed; GlaxoSmithKline Healthcare,
epithelial collar. The lumen of the cyst cavity was in direct continuity
B€uhl, Germany) for 15 minutes. After anesthesia, a full-thickness
with the root canal space through the major apical ramification
periosteal flap with 1 vertical incision was elevated. The MB root was
(Fig. 2C and G).
immediately visible (Fig. 1L), and the thin buccal bone in correspon-
The epithelial lining had the characteristics of stratified squamous
dence to the lesion was carefully removed (Fig. 1L) to improve the
epithelium. It was thick and irregular with ridges that branched to form
access to the apical periodontitis lesion (Fig. 1M). An attempt was
an arcading structure (Fig. 2C and D). The ridges of proliferating
made to obtain the resected root tip and the surrounding pathologic
epithelium enclosed islands of granulomatous tissue (Fig. 2D). These
soft tissue in their original relationship. The root tip was first resected
islands of connective tissue as well as the subepithelial connective tissue
approximately 3 mm short of the apex with a diamond bur cooled by
were well vascularized and infiltrated by many mononuclear inflamma-
sterile saline solution. Subsequently, the soft tissue was carefully
tory cells (Fig. 2D). Inflammation was milder toward the pseudocap-
enucleated from the bone crypt with smooth microelevators. The
sule, where only collagen bundles with some scattered chronic
cut surface of the MB root showed no signs of cracks, fractures, or
inflammatory cells could be observed. Epithelium was infiltrated by
accessory root canals at observation under 16 magnification
numerous polymorphonuclear leukocytes (Fig. 2E and F). The lumen
(Fig. 1N). The root canal filling material was removed 3-mm deep
of the cyst was apparently empty with some necrotic tissue debris and
with ultrasonic tips. MB1 and MB2 canals were joined in the last
blood remnants (Fig. 2C and G). Bacteria were not observed in the
3 mm in a single root-end cavity by using ultrasonic preparation
cyst lumen or on the cyst wall.
(Fig. 1O). After disinfection with 10% citric acid and 2% chlorhexidine,
In some sections, the main MB canal (MB1) in the very apical
the root-end cavity was filled with mineral trioxide aggregate (ProRoot
portion bifurcated into 2 symmetric ramifications whose lumina were
MTA; Dentsply, Konstanz, Germany) (Fig. 1P). The postoperative
completely filled by thick biofilms (Fig. 2H and I). The biofilm in
radiograph showed optimal root-end filling. Eleven months after
each ramification was clearly demarcated from the inflammatory
surgery, the tooth was asymptomatic, and a periapical radiograph
reaction and exhibited a certain amount of extracellular matrix in which
showed that healing was almost complete. The cavity was filled by newly
coccoidal forms dominated (Fig. 3A and B).
formed bone, and only residual widening of the periodontal ligament
The lumen of the large ramification communicating with the cyst
space around the MB root could be observed (Fig. 1Q).
cavity was also clogged by a thick bacterial biofilm with abundant
The patient gave consent for histologic examination. The biopsy
extracellular matrix, but bacterial filamentous forms were dominant
specimens, consisting of the root tip with the soft pathological tissue
in this location (Fig. 3C and D). Bacteria were also observed on the
(Fig. 2A and B) and 2 buccal cortical bone fragments, were immediately
walls of MB1 packed between the obturation material and the root canal
immersed in 10% neutral buffered formalin solution and sent to the
wall (Fig. 3E). Other sections disclosed additional ramifications, all
laboratory for histologic and histobacteriologic processing.
exhibiting bacterial colonization (Fig. 3F). Sections of the bone
fragments showed both marrow and compact bone with characteristics
of normality, except for some scattered osteoclasts (data not shown).
Tissue Processing
The specimens were kept in fixative for 5 days. Demineralization
was performed by immersion in an aqueous solution consisting of Discussion
a mixture of 22.5% (vol/vol) formic acid and 10% (wt/vol) sodium In the large majority of teeth with apical periodontitis, microbial
citrate for 3 weeks. The endpoint was determined radiographically. infection is present not only in the main root canal but also propagates
The specimens were washed in running water for 48 hours, dehydrated to variations of the internal anatomy of the system, including dentinal
in ascending grades of ethanol, cleared in xylene, infiltrated, and tubules, recesses, isthmuses, lateral canals, furcal canals, and apical
embedded in paraffin (melting point 56 C) according to standard ramifications (15–18). Bacterial biofilms are observed in the apical

1182 Arnold et al. JOE — Volume 39, Number 9, September 2013

 Case Report/Clinical Techniques

Figure 3. (A and B) A high-power view of the 2 apical ramifications. They are clogged with a thick biofilm in which round bacterial morphotypes dominate
(original magnification 400; Inset 1,000). (C) A detailed view of the left ramification in Figure 2H (original magnification 100). (D) Magnification of
the foraminal area showing a biofilm with abundant extracellular matrix in which filamentous forms are predominant (original magnification 400; inset
1,000). (E) A high-power view of the area from the left canal wall indicated by the arrow in Figure 2H. A bacterial biofilm can be observed between the
root canal wall and the obturation material (original magnification 100). (F) Another section: the exits of 4 ramifications can be discerned (original magnification
50).

part of the root canal system in about 80% of teeth with apical instruments and at an adequate WL (0.5 mm short), establishment
periodontitis (18). The older the infectious process, as inferred by and maintenance of apical foramen patency throughout the procedures,
lesion size or histopathologic diagnosis of a cyst, the more complex copious irrigation with highly concentrated (5%) NaOCl, smear layer
the infection in the apical canal is (18). Consequently, a complex removal, and a final rinse with and ultrasonic agitation of chlorhexidine.
infection lodged in a complex anatomy poses a great challenge to These approaches worked well for the other canals of the same tooth,
control and helps explain the lower success rate for teeth with apical leading to healing of the lesions on the distobuccal and palatal roots as
periodontitis when compared with teeth with no disease (19–21) revealed radiographically. The reason why the lesion on the MB root
and teeth with large lesions when compared with teeth with small persisted after treatment is probably a more complex anatomy of this
lesions (22–25). root, making single-visit disinfection less effective, but a definite
In this case report, bacteria persisting after treatment were establishment of the different tissue reaction cannot be established
arranged in biofilm structures located in an intricate network of apical because only the MB root was available for histopathologic analysis
ramifications that remained apparently unaffected by treatment. It is (for obvious ethical reasons).
noteworthy that treatment was performed based on optimal standards, It becomes clear that all these antimicrobial procedures, except
including apical preparation with reasonably large nickel-titanium for the patency files, were physically precluded from contacting the

JOE — Volume 39, Number 9, September 2013 Apical Delta Infection and Treatment Failure 1183
Case Report/Clinical Techniques
bacterial biofilms in the apical ramifications. The effects of the small 7. Ricucci D, Siqueira JF Jr. Fate of the tissue in lateral canals and apical ramifications
patency file, allegedly restricted to only 1 of the ramifications or the in response to pathologic conditions and treatment procedures. J Endod 2010;36:
1–15.
main apical foramen, were apparently negligible. In addition to keeping 8. Ricucci D, Siqueira JF Jr. Anatomic and microbiologic challenges to achieving
the apical foramen patent, patency files are also expected to disrupt success with endodontic treatment: a case report. J Endod 2008;34:1249–54.
apical biofilms mechanically (26) and chemically by carrying irrigant 9. Ricucci D, Siqueira JF Jr. Apical actinomycosis as a continuum of intraradicular and
solution to the very apical canal or improving delivery of irrigants to extraradicular infection: case report and critical review on its involvement with
that region (27). If these effects actually occurred to some degree, treatment failure. J Endod 2008;34:1124–9.
10. Nair PN, Sj€ogren U, Krey G, et al. Intraradicular bacteria and fungi in root-
histobacteriologic sections revealed that the apical biofilm apparently filled, asymptomatic human teeth with therapy-resistant periapical lesions:
recovered from such disturbances, at least morphologically, because a long-term light and electron microscopic follow-up study. J Endod 1990;
the biofilms seemed undisturbed in all ramifications. 16:580–8.
Treatment was performed in a single visit, and whether or not an 11. Carr GB, Schwartz RS, Schaudinn C, et al. Ultrastructural examination of failed molar
retreatment with secondary apical periodontitis: an examination of endodontic
interappointment medication would have improved prognosis can only biofilms in an endodontic retreatment failure. J Endod 2009;35:1303–9.
be speculated at this time. The main reason to apply a medication 12. Vieira AR, Siqueira JF Jr, Ricucci D, et al. Dentinal tubule infection as the cause of
between appointments of treatment of a tooth with apical periodontitis recurrent disease and late endodontic treatment failure: a case report. J Endod
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hydroxide paste) to diffuse and reach bacteria persisting unaffected 13. Taylor RD. Modification of the Brown and Brenn Gram stain for the differential
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15. Nair PNR. Light and electron microscopic studies of root canal flora and periapical
a complex case like the one reported here. This is because this alkaline lesions. J Endod 1987;13:29–39.
substance has low solubility, and as it diffuses through organic or 16. Siqueira JF Jr, R^oças IN, Lopes HP. Patterns of microbial colonization in primary
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1184 Arnold et al. JOE — Volume 39, Number 9, September 2013