Download Mycobacterium arupense infection in HIV

CASE REPORT
Mycobacterium arupense infection in HIV-infected patients
from Iran
P Heidarieh*, A Hashemi-Shahraki*†, A D Khosravi*†, S Zaker-Boustanabad‡, H Shojaei§
and M M Feizabadi**
*Department of Microbiology, School of Medicine, Alborz University of Medical Sciences, Karaj; †Infectious and Tropical Diseases Research
center, and Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz; ‡Parand Branch, Islamic
Azad University, Parand; §Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan; **Department of
Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Summary: Here we report two cases of infection caused by Mycobacterium arupense in HIV-infected patients who had received
Mycobacterium avium complex medication after primary treatment with antituberculous drugs. The causative agents were isolated
from the respiratory and blood specimens of the patients. The identification was based on conventional and molecular tests. Our
study provides further evidence on the role of this microorganism in clinical cases.
Keywords: Mycobacterium arupense, HIV, AIDS, atypical mycobacteria, non-tuberculous mycobacteria, treatment, Iran
INTRODUCTION
In the developed countries, presentations of HIV-related
pulmonary disease are changing from opportunistic infections
to chronic obstructive lung disease and pulmonary neoplasms
due to combination antiretroviral therapy (cART). However, opportunistic infections remain frequent in developing
countries as a result of limited access to cART.1
We describe here in the first report of Mycobacterium arupense
(M. arupense) infection in two cases of HIV-infected patients
who have received M. avium complex (MAC) medication after
primary treatment for tuberculosis.
CASE REPORTS
Case one
Strain AFP-0007 was isolated in pure culture from three different sputum samples and one broncho-alveolar lavage (BAL)
specimen of a 55-year-old Iranian HIV-positive man suffering
from chronic pulmonary disease in 2009.
The patient was hospitalized in 2004 due to fever, weight
loss, chronic chest pain and productive cough. The patient’s
tuberculin skin test was 6 mm in diameter and the chest X-ray
was normal. He had a past history of oral candidiasis with
HIV seropositivity and received antiretroviral therapy in 2002.
The initial laboratory examination of sputum samples by
Ziehl–Neelsen (ZN) staining revealed the presence of a few
coccobacillary acid-fast organisms which yielded pure culture
of M. tuberculosis, confirmed by IS6110 based-PCR. He was
successfully treated for pulmonary tuberculosis (TB) in 2004
with standard TB treatment including isoniazid, rifampicin,
Correspondence to: Azar Khosravi
Email: [email protected]
streptomycin and ethambutol for duration of nine months.
In spring 2009, the patient was hospitalized again in another
centre due to mild fever and chronic cough. Sputum samples
were evaluated for mycobacteria; all of the specimens were
culture positive for slowly growing Mycobacterium sp. Chest
X-ray was normal and tuberculin skin test was negative. The
patient was put on anti-TB treatment but, after a month,
direct examination of sputum was still positive. HIV status of
the patient showed a viral load of 800,000 copies/mL with 80
CD4 lymphocytes/mm3. Other laboratory indexes were: platelet count, 3 109/L; alanine aminotransferase level, 130 U/L;
alkaline phosphatase level, 400 U/L; albumin concentration,
20 g/L; globulin concentration, 15 g/L C-reactive protein
(CRP) up to 50 mg/dL and erythrocyte sedimentation rate
(ESR) up to 100 mm/hour.
Following primary identification of the isolated mycobacteria as non-tuberculous mycobacteria (NTM) by phenotypic
tests, to rule out any contamination a BAL specimen from
patient was examined, yielding a positive smear and culture
for the same isolate. The patient treatment regimen was
shifted from anti-TB drugs to clarithromycin (500 mg twice
daily), ethambutol (15 mg/kg daily) and rifabutin (150 mg/
day) due to a suspicion of M. avium complex (MAC) infection.
In addition, cART was started simultaneously with tenofovir/
emtricitabine and efavirenz for HIV treatment. The patient had
received 4 months of anti-TB treatment before switching to
MAC infection treatment.
Direct examination of sputum remained positive in follow-up
examination after a month, but the culture was found to be
negative. A subsequent follow-up examination after one year
revealed that both culture and direct smear were negative
and the patient was declared clinically and microbiologically
cured. The isolated Mycobacterium sp. (AFP-0007) was subsequently identified as M. arupense using sequence analysis.
The HIV status at the end of the M. arupense history was:
viral load 300,000 copies/mL with 400 CD4 lymphocytes/mm3.
DOI: 10.1177/0956462412472818. International Journal of STD & AIDS 2013; 24: 485–487
486
International Journal of STD & AIDS
Volume 24
June 2013
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Figure 1 Phylogenetic tree of nearly complete sequences of the 16S rRNA gene using the neighbour-joining method with Kimura’s twoparameter distance correction model. The tree shows the genetic relationship between strains AFP-0007 and AFP-0008 and other
Mycobacterium species. Nocardia asteroides ATCC 19247T was included as an outgroup. The percentages indicated at nodes represent
bootstrap levels supported by 1000 re-sampled data sets. The bars indicate sequence differences
Case two
MICROBIOLOGY
A 40-year-old male AIDS patient was hospitalized in January
2010 due to fever, weight loss, and cough for approximately
four weeks with simultaneous cytomegalovirus infection.
His past medical history included oesophageal candidiasis.
The patient had been receiving zidovudine/lamivudine
therapy since August 2009, when his CD4 count had dropped
(800 CD4 lymphocytes/mm3) and because of viral suppression
(HIV RNA 125,000 copies/mL), indinavir was also added to
patient’s regimen in October 2009.
On admission, he was severely immunocompromised (18
CD4 lymphocytes/mm3 and HIV RNA 1,000,000 copies/mL).
His laboratory tests showed an elevated CRP up to 43 mg/dL
and ESR up to 75 mm/hour. Other tests results were: platelet
count, 10 109/L; alanine aminotransferase level, 130 U/L;
alkaline phosphatase level, 220 U/L; albumin concentration,
20 g/L; globulin concentration, 11 g/L; CRP up to 43 mg/dL
and ESR up to 75 mm/hour. The patient’s tuberculin skin test
was negative and the chest X-ray was normal.
Slowly growing mycobacteria were recovered from two independent blood cultures from the patient in 2010. Combination
treatment of clarithromycin (500 mg twice daily), ethambutol
(15 mg/kg daily) and rifabutin (150 mg/day) were initially prescribed for treatment of disseminated MAC, while waiting for
later laboratory confirmation. A few weeks later, the patient
died due to liver failure. The isolated Mycobacterium sp. was
subsequently identified as M. arupense.
It is necessary to mention that the current work was
approved by the committees of medical ethics of Ahvaz
Jundishapour University of Medical Sciences (Ahvaz, Iran),
Imam Khomeini hospital (Tehran, Iran) and Masoud
Laboratory (Tehran, Iran).
Acid-fast organisms were detected in clinical specimens of case
one by ZN staining. The isolates from both cases grew rapidly
(5– 7 days) at 308C and slowly (10 –12 days) at 378C on
Lowenstein (LJ) medium. They had negative results for niacin
production, nitrate reduction, arylsulfatase activity (3-day),
urease production, iron uptake, tolerance to 5% NaCl and
growth on MacConkey agar without crystal violet. The isolates
also shown positive results for heat stable catalase, arylsulfatase
activity (14-day) and tween 80 hydrolysis.
Susceptibility testing was performed using in-house microdilution plates for slowly growing NTM. The results of susceptibility testing were as follows: Isolates were susceptible
to ethambutol (minimum inhibitory concentration [MIC]
0.25– 0.5 mg/mL), clarithromycin (MIC 0.5– 1 mg/mL) and rifabutin (MIC 0.25 mg/mL), but were resistant to rifampicin
(MIC .32 mg/mL), linezolid (MIC .128 mg/mL), streptomycin
(AFP-0007; MIC .32 mg/mL and AFP-0008; MIC .128 mg/
mL), ciprofloxacin (MIC .32 mg/mL) and moxifloxacin
(AFP-0007; MIC .32 mg/mL and AFP-0008; MIC .64 mg/mL).
Following DNA extraction, amplification and sequencing of
the nearly full length of 16S rRNA gene,2 partial segment
of hsp65 gene3 and rpoB gene,4 was carried out for each
isolate. The obtained sequences of 16S rRNA, hsp65 and rpoB
genes were aligned and compared with all existing sequences
of slowly growing mycobacteria retrieved from GenBank database using the jPhydit program.5
The identical 16S rRNA gene (1463 bp) sequences of the
strains AFP-0007 and AFP-0008 gave an identical sequence to
that of M. arupense ATCC BAA-1242T (GenBank accession No.
X93032). Figure 1 represents the neighbour-joining tree,6
based on 16S rRNA sequences of the strains AFP-0007 and
Heidarieh et al. Isolation of Mycobacterium arupense from Iran
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AFP-0008 matched sequences to that of M. arupense. The identical hsp65 gene (597 bp) and rpoB gene (710 bp) sequences
of the isolates also showed the identical sequence to that of
M. arupense ATCC DSM 44942T.
infection. Consequently, rapid molecular identification tests
are required for accurate identification of mycobacterial
species and control of related infections by proper antimicrobial
treatment strategies.
Nucleotide sequence accession numbers
ACKNOWLEDGEMENTS
The GenBank accession numbers of the 16S rRNA, hsp65
and rpoB gene for AFP-0007 as representative isolate are as
follows: JQ617910, JQ617911 and JQ617912.
DISCUSSION
In TB-endemic countries, particularly Iran, mycobacterial specimens are presumed to be TB by presence of acid fast bacilli in
smear microscopy and are not routinely cultured7 or screened
for NTM strains mainly due to lack of expertise methodology.
Consequently, the patients are started on treatment with antiTB drugs that have low activity or are inactive against infections
caused by other species of mycobacteria.8
M. arupense was introduced as novel species in 2006 as a
result of five-year isolation and identification of strains which
genotypically match to Mycobacterium sp. MCRO-6 from
65 human clinical samples.9 Tendovaginitis,10 tenosynovitis in
a patient with diabetes mellitus,11 flexor tenosynovitis in a
patient,12 pulmonary infection in malignant patient,13 osteomyelitis of the wrist,14 were reported due to infection
with M. arupense. To our knowledge, the present study is
the first report of pulmonary infections in two independent
HIV-infected patients in a developing country. Our patients
illustrated both the potential pathogenicity of M. arupense and
its occurrence in Iran. Repeated isolation of strains in pure
culture from a BAL specimen in Case one, as well as the
severely immunocompromised condition and isolation of
organism from blood samples in Case two, revealed that the
isolates were clinically significant and met American Thoracic
Society criteria.8
Due to the importance of isolation of M. arupense among HIV
patients in our study, conventional culture and identification
tests were performed and the antimycobacterial therapy was
undertaken based on susceptibility testing results for better
management of the infection. Conventional methods failed to
identify the clinical isolates at the species level, so, sequence
analysis of 16S rRNA gene, rpoB gene and hsp65 region was
carried out and definitive identity of the isolates was determined as M. arupense. The present report has highlighted the
problems in detection and identification of NTM in clinical
practice in developing countries like Iran, and misdiagnosis of
such infections as TB. Application of molecular methods such
as sequencing of the 16S rRNA, hsp65 and rpoB genes is
crucial for the accurate identification of such opportunistic
pathogens with ability to cause serious infections in immune
compromised individuals such as AIDS patients.
In conclusion, M. arupense can cause pulmonary infection in
immunocompromised patients such as those with HIV
The authors are grateful to research affairs, Ahvaz Jundishapur
University of Medical Sciences, Ahvaz, Iran, for financial
support (Grant No. 87101). Our appreciation goes to Director
General of the Masoud Laboratory (Tehran, Iran) and
Department of Infectious Diseases of Imam Khomeini
Hospital, Tehran, Iran, for providing the patients’ medical
records.
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