Download Rhodococcus - case study IJTLD

CASE STUDY
INT J TUBERC LUNG DIS 10(3):351–353
© 2006 The Union
Rhodococcus and Mycobacterium tuberculosis: masquerade
or mixed infection
N. F. Mistry,* Y. Dholakia,* D. T. B. D’Souza,* M. Taylor,† S. Hoffner,‡ T. J. Birdi*
* Foundation for Medical Research, Mumbai, India; † Imperial College School of Science, Technology and Medicine,
London, United Kingdom; ‡ Swedish Institute for Infectious Disease Control, Stockholm, Sweden
Q: “Although the” What word is missing?
SUMMARY
Rhodocci have a morphology similar to that of Mycobacterium tuberculosis (TB), and are indistinguishable
from normal diphtheroid flora. Symptoms include fever,
productive/non-productive cough and pleuritic chest
pain. Rhodococcal infections, being resistant to routine
anti-tuberculosis medications, may be misdiagnosed as
drug-resistant TB, thus prompting treatment for TB
with rifampicin-containing regimens that promote the
emergence of resistance. We present here a sputum smear
AFB-positive case who, although clinically cured, remains unresolved despite a series of technological investigations as to the cause of infection being purely rhodococci or mixed infection with M. tuberculosis.
K E Y W O R D S : Mycobacterium tuberculosis; Rhodococcus equi; rpo mutations; mixed infections; IS1081; Oxy R
ALTHOUGH they are resistant to routine antituberculosis treatment, rhodococci are morphologically similar to Mycobacterium tuberculosis (TB),
and are indistinguishable from normal diphtheroid
respiratory flora.1,2 Symptoms include fever, productive/
non-productive cough and pleuritic chest pain. Rhodococcal infections, being resistant to routine antituberculosis medications, may be misdiagnosed as
drug-resistant TB, thus prompting treatment for TB
with regimens that include rifampicin (RMP), which
promote the emergence of resistance.3
We present a sputum smear acid-fast bacilli (AFB)
positive case who, although cured, has remained unresolved as to whether the cause of infection is purely
rhodococci or mixed infection with M. tuberculosis.
attacks and the smear results, anti-tuberculosis treatment (HRZE*) was commenced on 18 May 2002
along with haematinics and ayurvedic immune modulators. The latter were temporarily withheld following a bout of acute bronchospasm. The paroxysmal
cough episodes persisted up to 9 weeks with fluctuations in respiratory symptoms. Drowsiness and fatigue attributed to isoniazid developed over time.
Weekly sputum samples were processed using the
modified Petroff’s method. The AFB count reduced
marginally until the third week after initiation of antituberculosis treatment. The AFB, although rod shaped,
appeared thicker, shorter and less curved in comparison to M. tuberculosis, and the presence of intracellular acid-fast cocci was also noted. Thereafter, the
count peaked at 2 months at 246 AFB/100 fields. All
routine mycobacterial growth media were culturenegative.
DNA analysis of the first sputum sample, conducted
at Swedish Bacteriological Laboratory, Stockholm,
using the Inno Lipa Kit (Innogenetics, Ghent, Belgium),
identified the organisms as M. tuberculosis complex,
positive for the rpo region, with the absence of band
S5 and the presence of a R5 band indicating RMP resistance. The same sample tested blind at Imperial
College, London, was also positive for rpo and
IS1081, an insertion sequence present in the M. tuberculosis complex. Oxy R sequencing also showed the
CASE REPORT
An 80-year-old male (weight 75 kg), resident of Mumbai and Pune, India, presented with insidious onset of
dry cough unresponsive to extensive supportive treatment in May 2002. With no other constitutional symptoms, clinical examination revealed only mild oropharyngeal congestion.
Routine investigations showed mild anaemia, reduced platelet count and lowered serum immunoglobulin G with normal chest X-ray. However, sputum
smears stained by Ziehl-Neelsen carbol fuchsin showed
50 AFB/100 fields. These were confirmed by another
reference laboratory.
Given the intractable cough leading to vasovagal
* H isoniazid; R rifampicin; Z pyrazinamide; E ethambutol.
Correspondence to: Nerges F Mistry, Foundation for Medical Research, 84-A, R G Thadani Marg, Worli Sea Face, Worli,
Mumbai 400 018, India. Tel: (91) 22 2493 4989/2876/8601. Fax: (91) 22 2493 2876. e-mail: [email protected]
Article submitted 6 April 2005. Final version accepted 20 September 2005.
352
The International Journal of Tuberculosis and Lung Disease
presence of nucleotide G at position 285 specific to M.
tuberculosis. Rpo gene sequencing showed the presence of two strains, of which the main one was M.
tuberculosis. On the basis of the above evidence, institution of second-line anti-tuberculosis treatment was
considered.
A thorough clinical, immunological and radiological (including chest high resolution computed tomography [HRCT]) examination was performed to facilitate a clinical decision 3 months post anti-tuberculosis
treatment. No abnormality was detected except for
chronic sinusitis of the left maxillary sinus. Fiberoptic
bronchoscopy revealed a normal tracheobronchial
tree. Processed bronchoalveolar aspirate was AFBpositive. The untreated sample was incubated at 37C in
chocolate blood agar, Saboraud’s agar and LöwensteinJensen as well as in Dubos broth and Mycobacteria
Growth Indicator Tubes (Becton Dickinson, Sparks,
MD, USA). While there was no growth of acid-fast
forms in routine mycobacterial growth media, colonies
obtained within 48 h on chocolate blood and Saborauds’ agar showed concurrent presence of acid-fast
cocci and short rods. On prolonged culture, however,
the colonies (2–4 mm in diameter, white, convex,
smooth and opaque) showed predominantly coccoid
forms. DNA analysis showed strong IS1081 bands
and were again positive for the rpo region. Sequence
analysis of this DNA using BLAST searches was positive for Rhodococcus equi-like organisms.
Anti-tuberculosis treatment was discontinued at
week 10 because of a stable clinical condition, lack of
radiological and bronchoscopic evidence of pulmonary TB and persistent side effects.
Two properties subsequently tested also conformed to those of the rhodococcus genus.4 Sputum
samples collected after August 2002 were negative for
AFB by the auromine O fluorescent method. Evidence
of a rhodococcal life cycle was noted when the coccoid colonies were inoculated into C57BL/6 mouse
peritoneal macrophages. Forty-eight hours post infection all intracellular organisms were noted to be AFB.
Rhodococci respond well to tetracycline, macrolides
and cotrimoxazole, but rapidly develop resistance to
routine anti-tuberculosis treatment.5 A prolonged sequential course of antibiotics (Figure) was prescribed
between August and October 2002. Symptoms gradually regressed, with normal lung function at 7 months
post onset. To date there is no recurrence of symptoms.
DISCUSSION
Rhodococci have recently emerged as opportunistic
pathogens and are sparsely reported.6 Infections in
both immunosuppressed and immunocompetent hosts
have been reported.7–9
The organism can be mistaken for M. tuberculosis on
routine AFB stain, but can be differentiated by its colony
characteristics. Rhodococci can also occur as mixed infections with M. tuberculosis, which might explain the
early detection of M. tuberculosis genes. Mycobacteria
and rhodococci are also known to have common antigens,10,11 and possibly identical or highly similar genomic sequences that lead to confounding results.
In an endemic area, the patient had probably been
pre-exposed to M. tuberculosis infection. With evidence of a multidrug-resistant M. tuberculosis strain
on the InnoLipa strip, it is doubtful that 3 months of
anti-tuberculosis treatment would have resolved the
infection even in a relatively well-nourished patient.
Although test positivity could have originated from
Figure Correlation between treatment, AFB counts and clinical status. Antibiotics given: 1) penicillin 15 days; 2) TMP SMX CLM 21 days; and 3) CPX CLM 5 days. AFB acid-fast bacilli; H isoniazid; R rifampicin; Z pyrazinamide; E ethambutol;
CT computed tomography; TMP trimethoprim; SMX sulfamethoxazole; CLM clarithromycin; CPX ciprofloxacin.
Rhodococcus and M. tuberculosis
DNA of bystander non-viable M. tuberculosis, the initial findings with sputum DNA suggest a mixed infection of M. tuberculosis and R. equi. However, the lack
of response to anti-tuberculosis treatment, the defervesence of clinical symptoms on prolonged non-anti-tuberculosis chemotherapy and the absence of symptoms on
follow-up is supportive of pure R. equi infection. Advanced age, mild immunodeficiency, chronic sinusitis
(constituting a persistent source of rhodococcus) along
with stress could have served as predisposing factors.
The gene analysis from the broncholavage sample
3.5 months post anti-tuberculosis treatment indicates
that rhodococcus, like M. tuberculosis, can be positive for IS1081 and rpo mutations. Common rpo
mutations have been reported in a variety of species.12
The kit manufacturer opined that it was ‘unlikely that
R. equi would react with the species-specific M.
tuberculosis probe if the strips were hybridised under
correct stringent conditions. Of course it cannot be
excluded completely’.
The question of single/mixed infection despite varied
investigations remains unresolved. It was only the absence of lung and bronchial lesions that prevented the
clinicians from embarking on a difficult second-line
regimen. That even gene-based technologies can yield
ambiguous results is a sobering thought for TB control programmes.
Acknowledgements
We are grateful to Dr C Rodrigues and Dr Z Udwadia of Hinduja
Hospital, Mumbai, for the bronchoscopy and subsequent investigations. We acknowledge the assistance of Air Marshal S K Dham,
Colonel K Lahiri (Armed Forces Medical College, Pune) and Dr J
Sorabji, Bombay Hospital, towards reaching a diagnosis. Dr H B
Singh, Ayurvedic consultant, Mumbai, prescribed the ayurvedic
immunomodulators.
353
References
1 Giacometti A, Cirioni O, Ancarani F, Del Prete MS, Fortuna
M, Scalise G. In vitro activities of polycationic peptides alone
and in combination with clinically used antimicrobial agents
against Rhodococcus equi. Antimicrob Agents Chemother
1999; 43: 2093–2096.
2 Takai S, Sasaki Y, Ikeda T, Uchida Y, Tsubaki S, Sekizaki T. Virulence of Rhodococcus equi isolates from patients with and
without AIDS. J Clin Microbiol 1994; 32: 457–460.
3 Gray K J, French N, Lugada E, Watera C, C F. Rhodococcus
equi and HIV infection in Uganda. J Infect 2000; 41: 227–231.
4 Cornish N, Washington J A. Rhodococcus equi infections: clinical features and laboratory diagnosis. Curr Clin topics Infect
Dis 1999; 19: 198–215.
5 Goodfellow M, Orchard V A. Antibiotic sensitivity of some
nocardioform bacteria and its value as a criterion for taxonomy. J Gen Microbiol 1974; 83: 375–387.
6 Prescott J F. Rhodococcus equi: an animal and human pathogen.
Clin Microbiol Rev 1991; 4: 20–34.
7 Clover D, Parks B, Harrell L J. Laboratory identification of
Rhodococcus equi from a patient with AIDS. Lab Med 1989;
20: 491–493.
8 Ebersole L L, Paturzo J L. Endophthalmitis caused by Rhodococcus equi Prescott serotype 4. J Clin Microbiol 1988; 26:
1221–1222.
9 La Rocca E, Gesu G, Caldara R, et al. Pulmonary infection
caused by Rhodococcus equi in a kidney and pancreas transplant
recipient: a case report. Transplantation 1998; 65: 1524–1525.
10 Ridell M. Studies on corynebacterial precipitinogens common
to mycobacteria, nocardia and rhodococcus. Int Archives Allergy Appl Immunol 1977; 55: 468–475.
11 Ridell M, Baker R, Lind A, Ouchterlong O. Immunodiffusion
studies of ribosomes in classification of mycobacteria and related
taxa. Int Archives Allergy Appl Immunol 1979; 59: 162–172.
12 Fines M, Pronost S, Maillard K, et al. Characterization of
mutations in the rpo gene associated with rifampicin resistance in Rhodococcus equi isolated from foals. J Clin Microbiol 2001; 39: 2784–2787.
RÉSUMÉ
Les rhodocoques ont une morphologie similaire à Mycobacterium tuberculosis (TB), et ne peuvent pas être distingués de la flore diphtéroïde normale. Les symptômes
comportent la fièvre, une toux productive ou non et des
douleurs thoraciques de type pleuritique. Les infections à
rhodocoques, étant résistantes aux médicaments antituberculeux de routine, peuvent être diagnostiquées par erreur comme TB à germes résistants, ce qui pousserait à un
traitement de la TB par des régimes incluant la rifampicine, facilitant l’apparition de la résistance. Nous présentons ici un cas à bacilloscopie positive des expectorations
qui, quoique cliniquement guéri, reste non résolu malgré
toute une série d’investigations technologiques concernant
la cause de l’infection, soit simplement des rhodocoques,
soit une infection mixte avec M. tuberculosis.
RESUMEN
Los rodococos poseen una morfología semejante a la de
Mycobacterium tuberculosis (TB) y son indistinguibles
de la flora difteroide normal. Los síntomas de la infección por rodococo son fiebre, tos seca o productiva y dolor torácico de tipo pleurítico. Esta infección es resistente a los medicamentos antituberculosos corrientes y
puede diagnosticarse erróneamente como TB farmacorresistente y llevar a la aplicación de una pauta terapéutica que incluya rifampicina, lo cual promueve la apari-
ción de resistencias. En el presente artículo se presenta el
caso de un paciente con examen microscópico del esputo
positivo para bacilos ácido-alcohol resistentes, curado
desde el punto de vista clínico y cuyo diagnóstico definitivo no pudo establecerse, pues una serie de estudios bacteriológicos y moleculares no permitió determinar si la infección era exclusivamente por rodococo o si se trataba de
una infección mixta por rodococo y por M. tuberculosis.