- Email: [email protected]
Detection of Mycobacterium tuberculosis Genome in Vitreous Fluid of Eyes with Multifocal Serpiginoid Choroiditis
Detection of Mycobacterium tuberculosis Genome in Vitreous Fluid of Eyes with Multifocal Serpiginoid Choroiditis Reema Bansal, MS,1,* Kusum Sharma, MD,2,* Amod Gupta, MS,1 Aman Sharma, MD,3 Mini P. Singh, MD,4 Vishali Gupta, MS,1 Samyak Mulkutkar, MS,1 Mohit Dogra, MS,1 Mangat R. Dogra, MS,1 Shivali Kamal, PhD,1 Surya Parkash Sharma, MSc,1 Paul D. Fiorella, PhD5 Purpose: To compare 3 different molecular techniques to detect the Mycobacterium tuberculosis genome in vitreous fluid of eyes with multifocal serpiginoid choroiditis (MSC). Design: Prospective, interventional case series. Participants: Eleven patients (11 eyes) with active MSC in at least 1 eye underwent diagnostic pars plana vitrectomy (PPV) between October 2012 and December 2013. Methods: Vitreous fluid samples were subjected to multitargeted polymerase chain reaction (PCR) for a M. tuberculosis assay, the Gene Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA), and a line probe assay (GenoType MTBDRplus; Hain Lifescience, GmbH, Nehren, Germany). The samples with positive results were subjected to rpoB gene sequencing to demonstrate rifampicin resistance. The clinical details, digital fundus imaging, and treatment details and outcomes also were noted. Main Outcome Measures: Detection of the M. tuberculosis genome and rifampicin resistance in the vitreous samples. Results: Of the 11 eyes subjected to PPV, the multitargeted PCR results for tuberculosis were positive for 10 eyes, the MTBDRplus assay results were positive in 6 eyes, and the Gene Xpert MTB/RIF assay results were positive in 4 eyes. Rifampicin resistance was detected in 3 eyes by rpoB gene sequencing, in 3 eyes by the MTBDRplus assay, and in 1 eye by the Gene Xpert MTB/RIF assay. Conclusions: We detected the M. tuberculosis genome in the vitreous fluid of eyes with MSC using 3 different molecular techniques. Rifampicin resistance was detected for the first time in eyes with MSC. Ophthalmology 2015;:1e11 ª 2015 by the American Academy of Ophthalmology.
Multifocal serpiginoid choroiditis (MSC) or serpiginous-like choroiditis is presumed to be tubercular in origin in tuberculosis-endemic areas.1e3 The diagnosis of MSC is essentially clinical, based on a characteristic morphologic presentation. It bears significant etiologic, demographic, clinical, therapeutic, and prognostic differences from classic serpiginous choroiditis.4e7 Although several infectious or noninfectious causes of MSC have been recognized,8 laboratory evidence for Mycobacterium tuberculosis or other causative agents is lacking. There are isolated reports of patients with serpiginous choroiditis with evidence of M. tuberculosis,2,9,10 herpes,11 or toxoplasmosis12 that has been shown using molecular techniques such as polymerase chain reaction (PCR). In tubercular MSC, 4-drug antitubercular therapy (ATT) in combination with corticosteroids prevents recurrences of inflammation.1e3 However, poor response in some patients, despite the conventional 4-drug therapy, suggests possible drug resistance.13 The role of diagnostic pars plana vitrectomy (PPV) in various uveitic entities is well known14,15; however, it has not been described in MSC. The intraocular specimen used 2015 by the American Academy of Ophthalmology Published by Elsevier Inc.
for PCR in serpiginous choroiditis in previous reports involved the aqueous humor2,11,12 more often than the vitreous humor.2,10 Although the vitreous has inflammatory cells in most eyes with tubercular MSC,1,4 the anterior chamber has minimal or no inflammation.1 We performed diagnostic PPV in MSC to detect the M. tuberculosis genome and drug resistance.
Methods We prospectively enrolled 11 patients (11 eyes) with active MSC in 1 or both eyes between October 2012 and December 2013 using following inclusion criteria: (1) evidence of active MSC lesions with central healing and active edges that showed early hypofluorescence and late hyperfluorescence on fluorescein angiography in at least 1 eye; (2) presence of significant vitreous cells (2þ or more) in the affected eye with or without anterior segment inflammation; (3) documented positive (10 mm of induration or more) tuberculin skin test (TST) results at 48 to 72 hours or QuantiFERON-TB Gold In-Tube (Cellestis Limited, Carnegie, Victoria, Australia) test results, or a strong clinical suspicion of MSC in the absence of any evidence of latent tuberculosis; and (4) all known causes of infectious uveitis except tuberculosis and http://dx.doi.org/10.1016/j.ophtha.2014.11.021 ISSN 0161-6420/14
1
Ophthalmology Volume -, Number -, Month 2015 known noninfectious uveitic syndromes excluded by clinical features or relevant tests. An induration of 10 mm or more was considered positive.16 Institute ethics committee approval was obtained. The study adhered to the tenets of the Declaration of Helsinki. To confirm our belief that vitrectomy has not been performed in MSC before our current study, we performed a MEDLINE search through PubMed (www.ncbi.nlm.nih.gov/pubmed) using the terms and medical subject heading terms serpiginouslike choroiditis, serpiginoid choroiditis, serpiginous choroiditis and polymerase chain reaction, or serpiginouslike choroiditis, serpiginoid choroiditis, serpiginous choroiditis, and vitrectomy.
Sample Collection After patients provided written informed consent, the affected eye underwent 23-gauge PPV by a single surgeon (A.G.). The first vitreous sample was collected in an undiluted state by flushing air, instead of fluid, through the infusion line. Using manual aspiration with a syringe connected to the aspiration line of the cutter, approximately 750 ml undiluted and 2 to 3 ml diluted vitreous samples were collected under direct visualization.
Molecular Assays The vitreous fluid samples were subjected to multitargeted PCR for M. tuberculosis,10 the Gene Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA), and a line probe assay (GenoType MTBDRplus; Hain Lifescience, GmbH, Nehren, Germany) for detecting the M. tuberculosis genome and rifampicin resistance (Fig 1). In multitargeted PCR, samples were considered positive if they showed 1 or more bands for the 3 genes tested. The samples with positive results for M. tuberculosis or rifampicin resistance by either of the techniques were subjected to rpoB gene sequencing to demonstrate rifampicin resistance.17 For the Gene Xpert MTB/RIF assay, at least 0.5 ml vitreous fluid was mixed with 2.5 ml buffer solution in a prelabeled, sterile, leak-proof Falcon (Tarsons Products Pvt Ltd, Kolkata, India) tube. After proper mixing and incubation at room temperature (as per the manufacturer’s instructions), the liquefied sample was transferred to the Xpert MTB/RIF cartridge, scanned, and loaded in to the instrument module. The reports were autogenerated by measuring fluorescent signals and embedded calculation algorithms in approximately 2 hours. The results were
interpreted as M. tuberculosis detected (low, medium, or high) or not detected, along with rifampicin resistance detected, not detected, or indeterminate. We performed the line probe assay using Genotype MTBDR plus version 2.0 kit. The procedure involved DNA isolation followed by multiplex amplification of targets with biotinylated primers, which underwent reverse hybridization with specific probes. The PCR amplification mix (45 ml) was prepared in a contamination-free room and DNA samples were added in a separate area. All the reagents required for multiplex PCR were included in the amplification mixes A and B and were optimized for the given test. It comprised a 2-step PCR analysis. The first step included 20 cycles of denaturation at 95 C for 30 seconds and annealing at 65 C for 2 minutes. The second step included 30 cycles of 95 C for 25 seconds, 50 C for 40 seconds, and extension at 70 C for 40 seconds. In addition, a 15-minute initial denaturation at 95 C and 8 minutes of extension at 72 C also were applied. The hybridization procedure was carried out manually using Twin Cubator (Hain Life Sciences) with a preinstalled hybridization protocol. Among the 21 test zones and 6 control zones in the MTBDRplus assay strip, the probes covered the rpoB gene for detecting rifampicin resistance and katG and inhA genes for detecting high-level and low-level isoniazid resistance, respectively. Positive staining results of all wild-type probes of a gene indicated no detectable mutation within the examined region. Absence of a signal for at least 1 wild-type probe indicated resistance of the tested strain to the respective drug. The samples from each eye also were subjected to multiplex PCR for virus and toxoplasmosis. The specific primers targeting the open reading frame 28 region of varicella zoster virus and the B1 gene of toxoplasma were used.18e20 Nested PCR for herpes simplex virus and cytomegalovirus was performed by amplifying the glycoprotein D gene and immediate early gene, respectively. The 5 ml eluted DNA was used for PCR amplification with 1X PCR buffer (10 mM Tris with 15 mM MgCl2), 200 mM deoxynucleotide, 1 mM of each primer, and 1 unit of Taq polymerase (Bangalore Genei, Bangalore, India). For the amplification of the second round for nested PCR, 5 ml amplified product from the first round was used. Appropriate positive and negative controls were included in each run. All necessary precautions were taken to prevent any carryover contamination. The amplicons were visualized by agarose gel electrophoresis after ethidium bromide staining.
Treatment
Figure 1. Schematic diagram showing how the vitreous sample was split between assays and what different assays were performed. CMV ¼ cytomegalovirus; HSV ¼ herpes simplex virus; PCR ¼ polymerase chain reaction; VZV ¼ varicella zoster virus.
2
The treatment was initiated with oral corticosteroids in all patients (prednisolone 0.5e1 mg/kg daily). The first-line 4-drug ATT included isoniazid (5 mg/kg daily), rifampicin (450 mg daily if body weight was less than 50 kg or 600 mg daily if body weight was 50 kg or more), ethambutol (15 mg/kg daily), and pyrazinamide (25e30 mg/kg daily) initially for 2 to 3 months under the supervision of the internist (A.S.). Thereafter, rifampicin and isoniazid were continued for another 9 to 10 months with pyridoxine supplementation. The treatment for multidrug-resistant (MDR) tuberculosis comprised pyrazinamide (1500 mg daily), levofloxacin (750 mg daily), ethionamide (750 mg daily), cycloserine (750 mg daily), and intramuscular injection of streptomycin 1 g daily for the first 5 months, followed by levofloxacin, ethionamide, and cycloserine for another 18 months. Liver function tests were carried out at baseline and follow-up to monitor for any drug toxicity to ATT. Topical corticosteroids and cycloplegics were added to patients with anterior segment inflammation. Immunosuppressive therapy consisting of azathioprine (2e2.5 mg/kg daily) was given to some patients showing
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 2. Gel documentation of multitargeted polymerase chain reaction results showing a 100-bp molecular marker (lane 1); positive control showing all 3 bands, that is, 123-bp IS6110, 240-bp MPB64, and 419-bp protein b (lane 2); clinical samples (of cases 1, 5, and 11) with positive results for all 3 bands (lane 3 to lane 5); clinical samples (of cases 3 and 10) with positive results for bands MPB64 and IS6110 (lane 6 and lane 7); and negative control (lane 8).
poor response to corticosteroids and ATT, under the internist’s supervision.
Results During the study period, 13 patients who met the inclusion criteria were offered the option of PPV. Of these, 2 patients declined. There were 8 males and 3 females. The mean age was 28.110.18 years (range, 15e50 years). The disease was bilateral in 5 patients. Nine patients had positive TST or QuantiFERON-TB Gold InTube test results. The multitargeted PCR results were positive in 10 eyes (Fig 2). Of the 10 eyes with positive PCR results, rpoB gene sequencing for rifampicin resistance detected mutations at codon 526 (case 1) and codon 531 (cases 5 and 11). In 6 eyes, the M. tuberculosis genome was detected by the MTBDRplus assay, of which rifampicin monoresistance was detected in 2 eyes (cases 1 and 11) and rifampicin plus isoniazid resistance was detected in 1 eye (case 5). The Gene Xpert assay showed positive results in 4 eyes and detected rifampicin resistance in 1 eye (case 1; Table 1). The patient with negative results by multitargeted PCR analysis also showed negative results by the Gene Xpert MTB/RIF assay, as well as the MTBDRplus assay, and showed no evidence of latent tuberculosis either (case 9). All the patients showed negative results for herpes simplex virus, varicella zoster virus, cytomegalovirus, and Toxoplasma on PCR analysis. All the patients received oral corticosteroids that were tapered according to the clinical response. Five among these also received the first-line 4-drug ATT, and 3 patients received ATT for MDR tuberculosis. Two patients with positive tuberculosis PCR results declined ATT (cases 6 and 7). One patient demonstrated significantly deranged liver function test results after 4-drug ATT (case 10) and subsequently was administered modified ATT comprising ethambutol (1000 mg daily) and levofloxacin (750 mg daily). Rifampicin (600 mg daily) and isoniazid (300 mg daily) were added after normalization of liver function test results. Ethambutol and levofloxacin were stopped after 3 and 4 months, respectively, with continuation of rifampicin and isoniazid. Four patients additionally
received immunosuppressive therapy for relentless progression despite corticosteroids and first-line ATT before diagnosing MDR tuberculosis (case 1) and for multiple recurrences despite corticosteroids (case 9) and first-line ATT (cases 10 and 11; Table 2). The mean follow up was 8.55.3 months (median, 7 months; range, 3e20 months). After starting the targeted therapy, 10 patients (all with positive multitargeted PCR results) showed resolution of inflammation, whereas 1 patient (with negative PCR results) had persistently active lesions despite corticosteroids and immunosuppressive therapy (case 9). A therapeutic trial of 4-drug ATT was added 6 months later, and the lesions have regressed. The best-corrected visual acuity (BCVA) ranged from counting fingers to 6/6 at the initial visit (6/12 or better in 5 eyes, 6/18e6/60 in 3 eyes, and less than 6/60 in 3 eyes). At the last visit, the BCVA was 6/12 or better in 6 eyes, between 6/18 and 6/60 in 3 eyes, and less than 6/60 in 2 eyes (Table 2).
Case Reports Case 1. A 21-year-old woman with active MSC in the right eye had decreased vision for 2 to 3 months. Her TST results were 2020 mm. She had been taking oral corticosteroids (prednisolone 1 mg/kg), prescribed elsewhere, for the previous 1 month with no improvement. At presentation to our department, her BCVA was 6/12 and 6/6 in the right and left eyes, respectively. The intraocular pressure was 12 mmHg in both eyes. The anterior segment was unremarkable in both eyes. The left eye fundus showed normal results. The right eye fundus showed active lesions of MSC in the posterior pole with pigmented scars peripherally (Fig 3A). We continued her oral corticosteroids in the same dose, but the lesions showed relentless progression at 1 week (Fig 3B), 3 weeks (Fig 3C), and 5 weeks (Fig 3D). Immunosuppressive therapy followed by first-line ATT was administered, but the lesions did not respond. The patient underwent a diagnostic PPV. Based on the positive results of PCR, the Gene Xpert assay, and the MDRTBplus assay, a definitive diagnosis of tuberculosis was established. The detection of rifampicin resistance by the Gene Xpert assay, the MDRTBplus assay, and its demonstration by rpoB gene sequencing (mutation at codon 526; Fig 3E) led to a revised diagnosis of MDR tuberculosis uveitis. Consequently, antieMDR tuberculosis therapy was started and the patient showed marked resolution of the lesions at 4 weeks (Fig 3F). At 8 months of follow-up, the lesions were healed (Fig 3G). Case 11. A 43-year-old man who had been a patient at our clinic for 11 years had active MSC in the right eye at initial presentation in 2003 and normal results in his left eye (Fig 4A). His TST results were positive and he received oral corticosteroids with conventional 4-drug ATT. The lesions in the right eye healed and he remained stable until 2007 (Fig 4B). In 2008, fresh lesions developed in the fellow eye in the macula (Fig 4C). The QuantiFERON-TB Gold In-Tube test results at this time were positive. He again received oral corticosteroids with conventional 4-drug ATT. Over the next 5 years, he showed remitting and relapsing lesions in the left eye (Fig 4DeG), for which he additionally received immunosuppressive therapy. After a recent recurrence (in 2014) in the left eye (Fig 4H), he underwent diagnostic PPV. The multitargeted PCR and MDRTBplus assay results were positive for tuberculosis, and the Gene Xpert assay results were negative. The MDRTBplus assay revealed rifampicin monoresistance, and the rpoB gene sequencing showed rifampicin resistance with mutation at 531 codon (Fig 4I). AntieMDR tuberculosis therapy was started, and the patient showed resolution of the lesions (Fig 4J). However, this patient demonstrated significant macular
3
4
Table 1. Demographic and Molecular Test Results of Patients Who Underwent Diagnostic Pars Plana Vitrectomy and Were Subjected to Multitargeted Polymerase Chain Reaction, Gene Xpert MTB/RIF Assay, and Line Probe Assay for Detecting Mycobacterium tuberculosis and Rifampicin Resistance Gene Xpert
Age (yrs)
1 2 3 4 5 6 7 8 9 10 11
21 23 27 30 15 50 22 30 22 26 33
Gender
Tuberculin Skin Test (mm)
QuantiFERON-TB Gold In-Tube Test Results
F M M M M F F M M M M
20 20 Necrotic 13 14 18 20 14 16 25 20 þ þ
ND þ ND ND þ ND ND þ þ
þ þ þ þ þ þ þ þ þ þ
Positive Bands IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 Negative IS6110 MPB64 IS6110 MPB64
Pab
Pab Pab Pab
Pab
rpoB Gene Sequencing
Mycobacterium tuberculosis Detected/Not Detected
Rifampicin Resistance or Sensitivity
Codon 526 RS RS RS Codon 531 RS RS RS ND RS Codon 531
þ Not Not Not þ Not þ þ Not Not Not
RR N/A N/A N/A RS N/A RS RS N/A N/A N/A
detected detected detected detected
detected detected detected
Line Probe Assay
Positive/Negative
Rifampicin Resistance or Sensitivity
Isoniazid Resistance or Sensitivity
þ þ þ þ þ þ
RR N/A N/A N/A RR N/A RS RS N/A RS RR
IS N/A N/A N/A IR N/A IS IS N/A IS IS
F ¼ female; IR ¼ isoniazid resistant; IS ¼ isoniazid sensitive; M ¼ male; N/A ¼ not applicable; ND ¼ not done; RR ¼ rifampicin resistant; RS ¼ rifampicin sensitive; þ ¼ positive; ¼ negative.
Table 2. Baseline Clinical, Treatment, and Outcome Details of Patients with Multifocal Serpiginoid Choroiditis Who Underwent Diagnostic Pars Plana Vitrectomy and Were Subjected to Multitargeted Polymerase Chain Reaction, Gene Xpert MTB/RIF Assay, and Line Probe Assay for Detecting Mycobacterium tuberculosis and Rifampicin Resistance
Case No.
Eye with Multifocal Serpiginoid Choroiditis
Initial Visual Acuity
1 2 3 4 5 6 7 8 9 10
Right Both Left Both Right Left Both Both Left Right
6/12 Counting fingers Counting fingers Counting fingers 6/9 6/60 6/6 6/24 6/24 6/9
CS CS CS CS CS CS CS CS CS CS
11
Both
6/6
CS þ azathioprine þ MDR TB treatment
Treatment þ þ þ þ þ
azathioprine þ MDR TB treatment ATT ATT ATT MDR TB treatment
þ ATT þ azathioprine; ATT (therapeutic trial) þ azathioprine þ modified ATT
Follow-up after Diagnostic Pars Plana Vitrectomy (mos)
Final Visual Acuity
Adverse Effects of Drugs
7 5 10 7.5 15 3 3 20 12 5
6/9 6/24 6/24 Counting fingers Counting fingers 6/12 6/12 6/6 6/18 6/9
None None None None None None None None None Highly deranged LFT results at 4 wks None
6.5
6/12
ATT ¼ antitubercular therapy (conventional first-line 4-drug therapy); CS ¼ corticosteroids; LFT ¼ liver function test; MDR TB ¼ multidrug-resistant tuberculosis.
Outcome Healed Healed; lost to follow-up Healed Healed Healed; significant cataract Healed; lost to follow-up Healed; lost to follow-up Healed Lesions healing Healed Healed
Ophthalmology Volume -, Number -, Month 2015
Case No.
Multitargeted Polymerase Chain Reaction Results
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 3.
5
Ophthalmology Volume -, Number -, Month 2015 involvement in the left eye that was uninvolved 10 years ago at the time of first presentation to us, owing to a marked diagnostic delay that caused multiple recurrences. At the last visit at 8 months after the initial visit, BCVA was 6/6 and 6/12 in the right and left eyes, respectively.
Discussion This is the first study of diagnostic PPV demonstrating the presence of M. tuberculosis DNA in the vitreous fluid of eyes with MSC. In this series, different molecular techniques established a definitive diagnosis of tuberculosis, such as multitargeted PCR analysis in 10 of 11 patients (previously reported sensitivity of 77.8%),10 the MTBDRplus assay in 6 cases, and the Gene Xpert MTB/ RIF assay in 4 cases. Multifocal serpiginous choroiditis presents as an isolated ocular disease, and definitive evidence of tuberculosis is extremely rare. The etiologic diagnosis is dependent on the clinical features, corroborative evidence, and response to ATT.1 Bhuibhar and Biswas21 substantiated a tubercular cause in a patient with amphigenous choroiditis by performing nested PCR analysis of an aqueous sample. Although most patients with MSC respond favorably to empirical treatment with 4-drug ATT and corticosteroids, a few patients fail to respond and pose a significant diagnostic and therapeutic challenge. Drug resistance, paradoxical worsening related to the toxic effects of ATT, poor compliance, or a nontubercular cause may be thought of as possible reasons in these patients. Although an invasive procedure, diagnostic PPV answers some of these questions. Emergence of drug resistance poses a major limitation in effectively controlling tuberculosis-related diseases, pulmonary or extrapulmonary. The conventional methods to detect resistance to rifampicin or isoniazid include culture of the mycobacteria and drug susceptibility testing, which are slow and time consuming. This leads to a delay in initiating appropriate therapy, allowing transmission and spread of MDR tuberculosis, particularly pulmonary tuberculosis. In the case of ocular involvement, this results in treatment failure, relapse, and significant visual morbidity resulting from uncontrolled intraocular inflammation. However, fear of overdiagnosing tuberculosis uveitis in an endemic region may lead to inadvertent use of corticosteroids and immunosuppressive agents. Therefore, in highprevalence countries, rapid diagnosis of tuberculosis is a prerequisite for administering targeted therapy to uveitis patients. Also, detection of MDR tuberculosis in these eyes by identifying resistance to rifampicin or isoniazid, as in other extrapulmonary sites, may save these eyes from blindness.
Significant advances in molecular techniques have offered the ability to obtain an early diagnosis of tuberculosis. In 2010, the World Health Organization approved the Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis.22 In addition to detecting M. tuberculosis DNA, it allows simultaneous testing of susceptibility to rifampicin (rifampicin) in a short time (approximately 2 hours). It is a closed cartridge system, based on a realtime heminested PCR that amplifies the rpoB gene 81-bp rifampicin resistanceedetermining region at codons 507 through 533. Several studies have reported high sensitivities and specificities from pulmonary and extrapulmonary samples.23e27 In our present series, the Gene Xpert assay could detect the M. tuberculosis genome in only 4 of 10 patients with positive PCR results, indicating a low sensitivity. The line probe assay (GenoType MTBDRplus) is another test in the rapid diagnostic algorithm of M. tuberculosis, allowing results within approximately 5 hours. In addition to detecting mutations associated with the rpoB gene for rifampicin resistance, the MTBDRplus assay detects katG and inhA gene mutations for high-level and low-level isoniazid resistance, respectively. It has a higher detection rate for rifampicin resistance compared with isoniazid resistance.28e30 In our study, the MTBDRplus assay detected M. tuberculosis in 6 eyes, of which rifampicin monoresistance was identified in 2 and combined rifampicin plus isoniazid resistance was detected in 1. On comparing the 3 tests for detecting M. tuberculosis in our patients, multitargeted PCR showed positive results in 10 of 11 eyes, the MTBDRplus assay showed positive results in 6 of 11 eyes, and the Gene Xpert assay showed positive results in 4 of 11 eyes. When comparing the performance of the 2 assays (Gene Xpert and MTBDRplus) for detecting MDR tuberculosis, the Gene Xpert assay detected rifampicin resistance in only 1 eye, whereas the MTBDRplus assay detected rifampicin resistance in 4 eyes (including 1 with isoniazid resistance). False-positive or overreporting of rifampicin resistance, which have been associated with the Gene Xpert assay,23 were not found in our study. On the contrary, the only patient detected by Gene Xpert as being resistant to rifampicin in our series was resistant to rifampicin by the MTBDRplus assay as well, as confirmed by rpoB gene sequencing to have a mutation at codon 526 (case 1). We found that although the Gene Xpert is preferable for faster detection of MDR tuberculosis (in approximately 2 hours), the MTBDRplus assay was superior in testing drug susceptibility. It additionally detected rifampicin monoresistance in 1 patient (case 11) who demonstrated positive PCR results but negative Gene Xpert results.
Figure 3. A, Fundus photograph of the right eye of a 21-year-old woman (case 1) with positive tuberculin skin test results showing active lesions of multifocal serpiginous choroiditis in the posterior pole along with healed lesions. B, Fundus photograph (left) and fluorescein angiograms (middle and right) showing progression of lesions 1 week after initial presentation. C and D, Fundus photographs showing relentless progression of lesions at (C) 3 and (D) 5 weeks, despite oral corticosteroids and antitubercular therapy. E, rpoB gene sequencing of vitreous fluid from case 1 showing a mutation at 526 codon (CAC [wild type]dTAC [mutant type]), within an 81-bp region of the rifampicin resistanceedetermining region of the rpoB gene. F and G, Fundus photographs obtained after diagnostic pars plana vitrectomy of the right eye and initiation of antiemultidrug-resistant tuberculosis therapy based on detection of rifampicin resistance; the patient showed marked resolution of lesions at (F) 4 weeks and (G) 8 months.
6
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 4. A, Fundus photographs from a 43-year-old man with positive tuberculin skin test results showing (left) active lesions of multifocal serpiginous choroiditis in the right eye at initial presentation in 2003 and (right) a normal left eye (right). B, Fundus photographs obtained after treatment with antitubercular therapy and corticosteroids. The lesions in the right eye healed and both eyes remained stable until 2007. C, Fundus photographs showing that although the lesions in the right eye remained scarred, fresh lesions developed in the fellow eye in the macula in 2008, for which retreatment with antitubercular therapy, corticosteroids, and immunosuppressive agents was administered. DeG, Fundus photographs of the left eye showing remitting and relapsing lesions during approximately the next 5 years: (D) 27, (E) 53, (F) 58, (G) and 62 months after the first appearance of lesions in the left eye. H, Montage fundus view and fluorescein angiogram of the left eye showing active lesions in the posterior pole as well as the periphery before performing diagnostic pars plana vitrectomy in the left eye. I, rpoB gene sequencing of vitreous fluid from case 11 showing a mutation at 531 codon (TCG [wild type]d TTG [mutant type]) within an 81-bp region of a rifampicin resistanceedetermining region of the rpoB gene. J, Fundus photograph of left eye 7 months after initiating antiemultidrug-resistant tuberculosis therapy showing healed lesions.
Because of an absence of overt systemic tuberculosis and lack of a histopathologic evidence in MSC, the exact pathogenesis remains unknown. The immune-mediated hypersensitivity reaction triggered by tuberculosis antigens has been one possible explanation.9 Although Rao et al9 demonstrated the presence of the M. tuberculosis within the retinal pigment epithelium (RPE) of a patient with panuveitis, our results demonstrate its presence in the
vitreous fluid of patients with active MSC and latent tuberculosis. Further, the role of RPE serving as a reservoir for intraocular M. tuberculosis infection recently was proposed by Nazari et al31 in human fetal RPE cells. The evidence of M. tuberculosis DNA in both of these intraocular sites (vitreous or RPE) reinforces the role of M. tuberculosis in causing or reactivating the inflammation of the inner choroid and RPE in MSC.
7
Ophthalmology Volume -, Number -, Month 2015
Figure 4. (continued).
8
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 4. (continued).
Evidence of tuberculosis in MSC is growing not only from endemic areas, but also from nonendemic areas, particularly in people migrating from endemic areas.4,8,32 We suggest looking for tuberculosis in these patients. Detection of rifampicin resistance in the present series led to revision of diagnosis to MDR tuberculosis and hence the treatment in 3 patients who showed poor response to initial therapy. Diagnostic PPV is associated with a decreased use of immunosuppressive therapy.33 Also, timely detection of rifampicin resistance can prevent inadvertent initiation of immunosuppressive drugs for possible paradoxical worsening of MSC. It emphasizes the need for a high index of suspicion of MDR tuberculosis in patients with MSC showing atypical features or poor response to therapy. Nazari et al31 have suggested the possibility of RPE cells as the site for latent tuberculosis as well as the site for reactivation of tubercular posterior uveitis. Negative results by tuberculosis PCR, Gene Xpert MTB/RIF assay, and MDRTBplus assay in 1 of our patients who also had no evidence of latent tuberculosis (patient 9) suggests the absence of local (ocular) or systemic M. tuberculosis antigenic stimulus in this patient and strengthens the causative association of latent tuberculosis with MSC in an endemic population. Although the beneficial effects of diagnostic PPV are well known in terms of adequate amount of vitreous sample for multiple tests,14,15,33 it has an added advantage over aqueous sampling in eyes with MSC because they usually have minimal or no inflammation in the anterior chamber.1
However, the potential adverse effects of routine vitreous surgery cannot be ignored, and only an experienced vitreoretinal surgeon should perform a diagnostic PPV, especially in eyes with good visual potential. Our study has a few limitations. We did not perform QuantiFERON-TB Gold In-Tube testing for all patients. Although this is a hot topic of discussion, and negative results effectively rule out tuberculosis, a substantial number of patients may have false-positive results.34 The MTBDRplus assay was performed on frozen aliquots, whereas the multitargeted PCR analysis and the Gene Xpert assay were performed on fresh samples. A possible bacterial disintegration could have impaired M. tuberculosis detection by the MTBDRplus assay and reduced the sensitivity of the test. This may have caused a discrepancy in the results. Furthermore, the comparison of laboratory (multitargeted PCR) and nonlaboratory (Gene Xpert and MTBDRplus assay) methods for detecting MDR tuberculosis in this study also is limited by a small sample size. Another limitation is a short follow-up. Therefore, long-term results of response to a targeted (rather than empirical) therapy, particularly MDR tuberculosis treatment, are not known. We conclude that in an endemic region the M. tuberculosis genome could be detected by different molecular techniques in eyes with MSC in patients who also had evidence of latent tuberculosis. A high index of suspicion should be maintained for rifampicin resistance in patients showing atypical features or poor response to initial conventional therapy.
9
Ophthalmology Volume -, Number -, Month 2015
References 1. Bansal R, Gupta A, Gupta V, et al. Tubercular serpiginouslike choroiditis presenting as multifocal serpiginoid choroiditis. Ophthalmology 2012;119:2334–42. 2. Gupta V, Gupta A, Arora S, et al. Presumed tubercular serpiginouslike choroiditis: clinical presentations and management. Ophthalmology 2003;110:1744–9. 3. Bansal R, Gupta A, Gupta V, et al. Role of anti-tubercular therapy in uveitis with latent/manifest tuberculosis. Am J Ophthalmol 2008;146:772–9. 4. Vasconcelos-Santos DV, Rao PK, Davies JB, et al. Clinical features of tubercular serpiginous like choroiditis in contrast to classic serpiginous choroiditis. Arch Ophthalmol 2010;128: 853–8. 5. Schatz H, Maumenee AE, Patz A. Geographic helicoid peripapillary choroidopathy. Trans Am Acad Ophthalmol Otolaryngol 1974;78:747–61. 6. Jabs DA, Rosenbaum JT, Foster CS, et al. Guidelines for the use of immunosuppressive drugs in patients with ocular inflammatory disorders: recommendations of an expert panel [review]. Am J Ophthalmol 2000;130:492–513. 7. Hooper PL, Kaplan HJ. Triple agent immunosuppression in serpiginous choroiditis. Ophthalmology 1991;98:944–51. 8. Khanamiri HN, Rao NA. Serpiginous choroiditis and infectious multifocal serpiginoid choroiditis. Surv Ophthalmol 2013;58:203–32. 9. Rao NA, Saraswathy S, Smith RE. Tuberculous uveitis: distribution of Mycobacterium tuberculosis in the retinal pigment epithelium. Arch Ophthalmol 2006;124:1777–9. 10. Sharma K, Gupta V, Bansal R, et al. Novel multi-targeted polymerase chain reaction for diagnosis of presumed tubercular uveitis. J Ophthalmic Inflamm Infect 2013;3:25. 11. Priya K, Madhavan HN, Reiser BJ, et al. Association of herpes-viruses in the aqueous humor of patients with serpiginous choroiditis: a polymerase chain reaction-based study. Ocul Immunol Inflamm 2002;10:253–61. 12. Mahendradas P, Kamath G, Mahalakshmi B, et al. Serpiginous choroiditis-like picture due to ocular toxoplasmosis. Ocul Immunol Inflamm 2007;15:127–30. 13. Sharma K, Bansal R, Sharma A, et al. Successful treatment of rifampicin resistant intraocular tuberculosis. Ocul Immunol Inflamm 2014 Mar 21. [Epub ahead of print]. 14. Mruthyunjaya P, Jumper JM, McCallum R, et al. Diagnostic yield of vitrectomy in eyes with suspected posterior segment infection or malignancy. Ophthalmology 2002;109:1123–9. 15. Davis JL, Miller DM, Ruiz P. Diagnostic testing of vitrectomy specimens. Am J Ophthalmol 2005;140:822–9. 16. American Thoracic Society. Diagnostic standards and classification of tuberculosis and other mycobacterial diseases (14th ed). Am Rev Respir Dis 1981;123:343–58. 17. Taniguchi H, Aramaki H, Nikaido Y, et al. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol Lett 1996;144:103–8. 18. Read SJ, Kurtz JB. Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay. J Clin Microbiol 1999;37: 1352–5.
10
19. Zhang Y, Kimura T, Fujiki K, et al. Multiplex polymerase chain reaction for detection of herpes simplex virus type 1, type 2, cytomegalovirus, and varicellazoster virus in ocular viral infections. Jpn J Ophthalmol 2003;47:260–4. 20. Mahalakshmi B, Therese KL, Shyamala G, et al. Toxoplasma gondii detection by nested polymerase chain reaction in lens aspirate and peripheral blood leukocyte in congenital cataract patients: the first report from a tertiary eye hospital in India. Curr Eye Res 2007;32:653–7. 21. Bhuibhar SS, Biswas J. Nested PCR-positive tubercular ampiginous choroiditis: a case report. Ocul Immunol Inflamm 2012;20:303–5. 22. World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system. Policy statement. Geneva, Switzerland: World Health Organization; 2011. Available at: http://apps.who.int/iris/ handle/10665/44586. Accessed on 22nd July, 2014. 23. Boehme CC, Nabeta P, Hillemann D, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005–15. 24. Marlowke EM, Novak Weekley SM, Cumpio J, et al. Evaluation of the Cepheid Xpert MTB/RIF assay for the direct detection of Mycobacterium tuberculosis complex from respiratory specimens. J Clin Microbiol 2011;49:1621–3. 25. Hillemann D, Ruesch-Gerdes S, Boehme C, Richter E. Rapid molecular detection of extrapulmonary tuberculosis by automated GeneXpert MTB/RIF system. J Clin Microbiol 2011;49:1202–5. 26. Nhu NTQ, Heemskerk D, Thu DDA, et al. Evaluation of GeneXpert MTB/RIF for diagnosis of tuberculous meningitis. J Clin Microbiol 2014;52:226–33. 27. Lawn SD, Nicol MP. Xpert MTB/RIF assay: development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin resistance. Future Microbiol 2011;6:1067–82. 28. Ling D, Zwerling A, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta-analysis. Eur Respir J 2008;32:1165–74. 29. Albert H, Bwanga F, Mukkada S, et al. Rapid screening of MDR-TB using molecular line probe assay is feasible in Uganda. BMC Infect Dis 2010;10:41. 30. Farooqi JQ, Khan E, Alam SM, et al. Line probe assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan. J Pak Med Assoc 2012;62:767–72. 31. Nazari H, Karakousis PC, Rao NA. Replication of Mycobacterium tuberculosis in retinal pigment epithelium. JAMA Ophthalmol 2014;132:724–9. 32. Gan WL, Jones NP. Serpiginous-like choroiditis as a marker for tuberculosis in a non-endemic area. Br J Ophthalmol 2013;97:644–7. 33. Oahalou A, Schellekens PAWJF, De Groot-Mijnes JD, Rothova A. Diagnostic pars plana vitrectomy and aqueous analyses in patients with uveitis of unknown cause. Retina 2014;34:108–14. 34. Pepple KL, VanGelder R, Forooghian F. Caveats about QuantiFERON-TB Gold In-Tube testing for uveitis. Am J Ophthalmol 2014;157:752–3.
Bansal et al
Diagnostic Vitrectomy in MSC
Footnotes and Financial Disclosures Originally received: August 27, 2014. Final revision: November 18, 2014. Accepted: November 18, 2014. Available online: ---.
*Both Dr. Bansal and Dr. Sharma contributed equally as first authors.
Manuscript no. 2014-1384.
1
Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India. 2 Department of Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India. 3
Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
4
Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
5
Bureau of Health Laboratories, Jacksonville, Florida.
Presented as a poster at: American Academy of Ophthalmology Annual Meeting, October 2014, Chicago, Illinois.
Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, India. Abbreviations and Acronyms: ATT ¼ antitubercular therapy; BCVA ¼ best-corrected visual acuity; MDR ¼ multidrug-resistant; MSC ¼ multifocal serpiginoid choroiditis; PCR ¼ polymerase chain reaction; PPV ¼ pars plana vitrectomy; RPE ¼ retinal pigment epithelium; TST ¼ tuberculin skin test. Correspondence: Amod Gupta, MS, Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India 160012. E-mail: [email protected].
11
Multifocal serpiginoid choroiditis (MSC) or serpiginous-like choroiditis is presumed to be tubercular in origin in tuberculosis-endemic areas.1e3 The diagnosis of MSC is essentially clinical, based on a characteristic morphologic presentation. It bears significant etiologic, demographic, clinical, therapeutic, and prognostic differences from classic serpiginous choroiditis.4e7 Although several infectious or noninfectious causes of MSC have been recognized,8 laboratory evidence for Mycobacterium tuberculosis or other causative agents is lacking. There are isolated reports of patients with serpiginous choroiditis with evidence of M. tuberculosis,2,9,10 herpes,11 or toxoplasmosis12 that has been shown using molecular techniques such as polymerase chain reaction (PCR). In tubercular MSC, 4-drug antitubercular therapy (ATT) in combination with corticosteroids prevents recurrences of inflammation.1e3 However, poor response in some patients, despite the conventional 4-drug therapy, suggests possible drug resistance.13 The role of diagnostic pars plana vitrectomy (PPV) in various uveitic entities is well known14,15; however, it has not been described in MSC. The intraocular specimen used 2015 by the American Academy of Ophthalmology Published by Elsevier Inc.
for PCR in serpiginous choroiditis in previous reports involved the aqueous humor2,11,12 more often than the vitreous humor.2,10 Although the vitreous has inflammatory cells in most eyes with tubercular MSC,1,4 the anterior chamber has minimal or no inflammation.1 We performed diagnostic PPV in MSC to detect the M. tuberculosis genome and drug resistance.
Methods We prospectively enrolled 11 patients (11 eyes) with active MSC in 1 or both eyes between October 2012 and December 2013 using following inclusion criteria: (1) evidence of active MSC lesions with central healing and active edges that showed early hypofluorescence and late hyperfluorescence on fluorescein angiography in at least 1 eye; (2) presence of significant vitreous cells (2þ or more) in the affected eye with or without anterior segment inflammation; (3) documented positive (10 mm of induration or more) tuberculin skin test (TST) results at 48 to 72 hours or QuantiFERON-TB Gold In-Tube (Cellestis Limited, Carnegie, Victoria, Australia) test results, or a strong clinical suspicion of MSC in the absence of any evidence of latent tuberculosis; and (4) all known causes of infectious uveitis except tuberculosis and http://dx.doi.org/10.1016/j.ophtha.2014.11.021 ISSN 0161-6420/14
1
Ophthalmology Volume -, Number -, Month 2015 known noninfectious uveitic syndromes excluded by clinical features or relevant tests. An induration of 10 mm or more was considered positive.16 Institute ethics committee approval was obtained. The study adhered to the tenets of the Declaration of Helsinki. To confirm our belief that vitrectomy has not been performed in MSC before our current study, we performed a MEDLINE search through PubMed (www.ncbi.nlm.nih.gov/pubmed) using the terms and medical subject heading terms serpiginouslike choroiditis, serpiginoid choroiditis, serpiginous choroiditis and polymerase chain reaction, or serpiginouslike choroiditis, serpiginoid choroiditis, serpiginous choroiditis, and vitrectomy.
Sample Collection After patients provided written informed consent, the affected eye underwent 23-gauge PPV by a single surgeon (A.G.). The first vitreous sample was collected in an undiluted state by flushing air, instead of fluid, through the infusion line. Using manual aspiration with a syringe connected to the aspiration line of the cutter, approximately 750 ml undiluted and 2 to 3 ml diluted vitreous samples were collected under direct visualization.
Molecular Assays The vitreous fluid samples were subjected to multitargeted PCR for M. tuberculosis,10 the Gene Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA), and a line probe assay (GenoType MTBDRplus; Hain Lifescience, GmbH, Nehren, Germany) for detecting the M. tuberculosis genome and rifampicin resistance (Fig 1). In multitargeted PCR, samples were considered positive if they showed 1 or more bands for the 3 genes tested. The samples with positive results for M. tuberculosis or rifampicin resistance by either of the techniques were subjected to rpoB gene sequencing to demonstrate rifampicin resistance.17 For the Gene Xpert MTB/RIF assay, at least 0.5 ml vitreous fluid was mixed with 2.5 ml buffer solution in a prelabeled, sterile, leak-proof Falcon (Tarsons Products Pvt Ltd, Kolkata, India) tube. After proper mixing and incubation at room temperature (as per the manufacturer’s instructions), the liquefied sample was transferred to the Xpert MTB/RIF cartridge, scanned, and loaded in to the instrument module. The reports were autogenerated by measuring fluorescent signals and embedded calculation algorithms in approximately 2 hours. The results were
interpreted as M. tuberculosis detected (low, medium, or high) or not detected, along with rifampicin resistance detected, not detected, or indeterminate. We performed the line probe assay using Genotype MTBDR plus version 2.0 kit. The procedure involved DNA isolation followed by multiplex amplification of targets with biotinylated primers, which underwent reverse hybridization with specific probes. The PCR amplification mix (45 ml) was prepared in a contamination-free room and DNA samples were added in a separate area. All the reagents required for multiplex PCR were included in the amplification mixes A and B and were optimized for the given test. It comprised a 2-step PCR analysis. The first step included 20 cycles of denaturation at 95 C for 30 seconds and annealing at 65 C for 2 minutes. The second step included 30 cycles of 95 C for 25 seconds, 50 C for 40 seconds, and extension at 70 C for 40 seconds. In addition, a 15-minute initial denaturation at 95 C and 8 minutes of extension at 72 C also were applied. The hybridization procedure was carried out manually using Twin Cubator (Hain Life Sciences) with a preinstalled hybridization protocol. Among the 21 test zones and 6 control zones in the MTBDRplus assay strip, the probes covered the rpoB gene for detecting rifampicin resistance and katG and inhA genes for detecting high-level and low-level isoniazid resistance, respectively. Positive staining results of all wild-type probes of a gene indicated no detectable mutation within the examined region. Absence of a signal for at least 1 wild-type probe indicated resistance of the tested strain to the respective drug. The samples from each eye also were subjected to multiplex PCR for virus and toxoplasmosis. The specific primers targeting the open reading frame 28 region of varicella zoster virus and the B1 gene of toxoplasma were used.18e20 Nested PCR for herpes simplex virus and cytomegalovirus was performed by amplifying the glycoprotein D gene and immediate early gene, respectively. The 5 ml eluted DNA was used for PCR amplification with 1X PCR buffer (10 mM Tris with 15 mM MgCl2), 200 mM deoxynucleotide, 1 mM of each primer, and 1 unit of Taq polymerase (Bangalore Genei, Bangalore, India). For the amplification of the second round for nested PCR, 5 ml amplified product from the first round was used. Appropriate positive and negative controls were included in each run. All necessary precautions were taken to prevent any carryover contamination. The amplicons were visualized by agarose gel electrophoresis after ethidium bromide staining.
Treatment
Figure 1. Schematic diagram showing how the vitreous sample was split between assays and what different assays were performed. CMV ¼ cytomegalovirus; HSV ¼ herpes simplex virus; PCR ¼ polymerase chain reaction; VZV ¼ varicella zoster virus.
2
The treatment was initiated with oral corticosteroids in all patients (prednisolone 0.5e1 mg/kg daily). The first-line 4-drug ATT included isoniazid (5 mg/kg daily), rifampicin (450 mg daily if body weight was less than 50 kg or 600 mg daily if body weight was 50 kg or more), ethambutol (15 mg/kg daily), and pyrazinamide (25e30 mg/kg daily) initially for 2 to 3 months under the supervision of the internist (A.S.). Thereafter, rifampicin and isoniazid were continued for another 9 to 10 months with pyridoxine supplementation. The treatment for multidrug-resistant (MDR) tuberculosis comprised pyrazinamide (1500 mg daily), levofloxacin (750 mg daily), ethionamide (750 mg daily), cycloserine (750 mg daily), and intramuscular injection of streptomycin 1 g daily for the first 5 months, followed by levofloxacin, ethionamide, and cycloserine for another 18 months. Liver function tests were carried out at baseline and follow-up to monitor for any drug toxicity to ATT. Topical corticosteroids and cycloplegics were added to patients with anterior segment inflammation. Immunosuppressive therapy consisting of azathioprine (2e2.5 mg/kg daily) was given to some patients showing
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 2. Gel documentation of multitargeted polymerase chain reaction results showing a 100-bp molecular marker (lane 1); positive control showing all 3 bands, that is, 123-bp IS6110, 240-bp MPB64, and 419-bp protein b (lane 2); clinical samples (of cases 1, 5, and 11) with positive results for all 3 bands (lane 3 to lane 5); clinical samples (of cases 3 and 10) with positive results for bands MPB64 and IS6110 (lane 6 and lane 7); and negative control (lane 8).
poor response to corticosteroids and ATT, under the internist’s supervision.
Results During the study period, 13 patients who met the inclusion criteria were offered the option of PPV. Of these, 2 patients declined. There were 8 males and 3 females. The mean age was 28.110.18 years (range, 15e50 years). The disease was bilateral in 5 patients. Nine patients had positive TST or QuantiFERON-TB Gold InTube test results. The multitargeted PCR results were positive in 10 eyes (Fig 2). Of the 10 eyes with positive PCR results, rpoB gene sequencing for rifampicin resistance detected mutations at codon 526 (case 1) and codon 531 (cases 5 and 11). In 6 eyes, the M. tuberculosis genome was detected by the MTBDRplus assay, of which rifampicin monoresistance was detected in 2 eyes (cases 1 and 11) and rifampicin plus isoniazid resistance was detected in 1 eye (case 5). The Gene Xpert assay showed positive results in 4 eyes and detected rifampicin resistance in 1 eye (case 1; Table 1). The patient with negative results by multitargeted PCR analysis also showed negative results by the Gene Xpert MTB/RIF assay, as well as the MTBDRplus assay, and showed no evidence of latent tuberculosis either (case 9). All the patients showed negative results for herpes simplex virus, varicella zoster virus, cytomegalovirus, and Toxoplasma on PCR analysis. All the patients received oral corticosteroids that were tapered according to the clinical response. Five among these also received the first-line 4-drug ATT, and 3 patients received ATT for MDR tuberculosis. Two patients with positive tuberculosis PCR results declined ATT (cases 6 and 7). One patient demonstrated significantly deranged liver function test results after 4-drug ATT (case 10) and subsequently was administered modified ATT comprising ethambutol (1000 mg daily) and levofloxacin (750 mg daily). Rifampicin (600 mg daily) and isoniazid (300 mg daily) were added after normalization of liver function test results. Ethambutol and levofloxacin were stopped after 3 and 4 months, respectively, with continuation of rifampicin and isoniazid. Four patients additionally
received immunosuppressive therapy for relentless progression despite corticosteroids and first-line ATT before diagnosing MDR tuberculosis (case 1) and for multiple recurrences despite corticosteroids (case 9) and first-line ATT (cases 10 and 11; Table 2). The mean follow up was 8.55.3 months (median, 7 months; range, 3e20 months). After starting the targeted therapy, 10 patients (all with positive multitargeted PCR results) showed resolution of inflammation, whereas 1 patient (with negative PCR results) had persistently active lesions despite corticosteroids and immunosuppressive therapy (case 9). A therapeutic trial of 4-drug ATT was added 6 months later, and the lesions have regressed. The best-corrected visual acuity (BCVA) ranged from counting fingers to 6/6 at the initial visit (6/12 or better in 5 eyes, 6/18e6/60 in 3 eyes, and less than 6/60 in 3 eyes). At the last visit, the BCVA was 6/12 or better in 6 eyes, between 6/18 and 6/60 in 3 eyes, and less than 6/60 in 2 eyes (Table 2).
Case Reports Case 1. A 21-year-old woman with active MSC in the right eye had decreased vision for 2 to 3 months. Her TST results were 2020 mm. She had been taking oral corticosteroids (prednisolone 1 mg/kg), prescribed elsewhere, for the previous 1 month with no improvement. At presentation to our department, her BCVA was 6/12 and 6/6 in the right and left eyes, respectively. The intraocular pressure was 12 mmHg in both eyes. The anterior segment was unremarkable in both eyes. The left eye fundus showed normal results. The right eye fundus showed active lesions of MSC in the posterior pole with pigmented scars peripherally (Fig 3A). We continued her oral corticosteroids in the same dose, but the lesions showed relentless progression at 1 week (Fig 3B), 3 weeks (Fig 3C), and 5 weeks (Fig 3D). Immunosuppressive therapy followed by first-line ATT was administered, but the lesions did not respond. The patient underwent a diagnostic PPV. Based on the positive results of PCR, the Gene Xpert assay, and the MDRTBplus assay, a definitive diagnosis of tuberculosis was established. The detection of rifampicin resistance by the Gene Xpert assay, the MDRTBplus assay, and its demonstration by rpoB gene sequencing (mutation at codon 526; Fig 3E) led to a revised diagnosis of MDR tuberculosis uveitis. Consequently, antieMDR tuberculosis therapy was started and the patient showed marked resolution of the lesions at 4 weeks (Fig 3F). At 8 months of follow-up, the lesions were healed (Fig 3G). Case 11. A 43-year-old man who had been a patient at our clinic for 11 years had active MSC in the right eye at initial presentation in 2003 and normal results in his left eye (Fig 4A). His TST results were positive and he received oral corticosteroids with conventional 4-drug ATT. The lesions in the right eye healed and he remained stable until 2007 (Fig 4B). In 2008, fresh lesions developed in the fellow eye in the macula (Fig 4C). The QuantiFERON-TB Gold In-Tube test results at this time were positive. He again received oral corticosteroids with conventional 4-drug ATT. Over the next 5 years, he showed remitting and relapsing lesions in the left eye (Fig 4DeG), for which he additionally received immunosuppressive therapy. After a recent recurrence (in 2014) in the left eye (Fig 4H), he underwent diagnostic PPV. The multitargeted PCR and MDRTBplus assay results were positive for tuberculosis, and the Gene Xpert assay results were negative. The MDRTBplus assay revealed rifampicin monoresistance, and the rpoB gene sequencing showed rifampicin resistance with mutation at 531 codon (Fig 4I). AntieMDR tuberculosis therapy was started, and the patient showed resolution of the lesions (Fig 4J). However, this patient demonstrated significant macular
3
4
Table 1. Demographic and Molecular Test Results of Patients Who Underwent Diagnostic Pars Plana Vitrectomy and Were Subjected to Multitargeted Polymerase Chain Reaction, Gene Xpert MTB/RIF Assay, and Line Probe Assay for Detecting Mycobacterium tuberculosis and Rifampicin Resistance Gene Xpert
Age (yrs)
1 2 3 4 5 6 7 8 9 10 11
21 23 27 30 15 50 22 30 22 26 33
Gender
Tuberculin Skin Test (mm)
QuantiFERON-TB Gold In-Tube Test Results
F M M M M F F M M M M
20 20 Necrotic 13 14 18 20 14 16 25 20 þ þ
ND þ ND ND þ ND ND þ þ
þ þ þ þ þ þ þ þ þ þ
Positive Bands IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 IS6110 MPB64 Negative IS6110 MPB64 IS6110 MPB64
Pab
Pab Pab Pab
Pab
rpoB Gene Sequencing
Mycobacterium tuberculosis Detected/Not Detected
Rifampicin Resistance or Sensitivity
Codon 526 RS RS RS Codon 531 RS RS RS ND RS Codon 531
þ Not Not Not þ Not þ þ Not Not Not
RR N/A N/A N/A RS N/A RS RS N/A N/A N/A
detected detected detected detected
detected detected detected
Line Probe Assay
Positive/Negative
Rifampicin Resistance or Sensitivity
Isoniazid Resistance or Sensitivity
þ þ þ þ þ þ
RR N/A N/A N/A RR N/A RS RS N/A RS RR
IS N/A N/A N/A IR N/A IS IS N/A IS IS
F ¼ female; IR ¼ isoniazid resistant; IS ¼ isoniazid sensitive; M ¼ male; N/A ¼ not applicable; ND ¼ not done; RR ¼ rifampicin resistant; RS ¼ rifampicin sensitive; þ ¼ positive; ¼ negative.
Table 2. Baseline Clinical, Treatment, and Outcome Details of Patients with Multifocal Serpiginoid Choroiditis Who Underwent Diagnostic Pars Plana Vitrectomy and Were Subjected to Multitargeted Polymerase Chain Reaction, Gene Xpert MTB/RIF Assay, and Line Probe Assay for Detecting Mycobacterium tuberculosis and Rifampicin Resistance
Case No.
Eye with Multifocal Serpiginoid Choroiditis
Initial Visual Acuity
1 2 3 4 5 6 7 8 9 10
Right Both Left Both Right Left Both Both Left Right
6/12 Counting fingers Counting fingers Counting fingers 6/9 6/60 6/6 6/24 6/24 6/9
CS CS CS CS CS CS CS CS CS CS
11
Both
6/6
CS þ azathioprine þ MDR TB treatment
Treatment þ þ þ þ þ
azathioprine þ MDR TB treatment ATT ATT ATT MDR TB treatment
þ ATT þ azathioprine; ATT (therapeutic trial) þ azathioprine þ modified ATT
Follow-up after Diagnostic Pars Plana Vitrectomy (mos)
Final Visual Acuity
Adverse Effects of Drugs
7 5 10 7.5 15 3 3 20 12 5
6/9 6/24 6/24 Counting fingers Counting fingers 6/12 6/12 6/6 6/18 6/9
None None None None None None None None None Highly deranged LFT results at 4 wks None
6.5
6/12
ATT ¼ antitubercular therapy (conventional first-line 4-drug therapy); CS ¼ corticosteroids; LFT ¼ liver function test; MDR TB ¼ multidrug-resistant tuberculosis.
Outcome Healed Healed; lost to follow-up Healed Healed Healed; significant cataract Healed; lost to follow-up Healed; lost to follow-up Healed Lesions healing Healed Healed
Ophthalmology Volume -, Number -, Month 2015
Case No.
Multitargeted Polymerase Chain Reaction Results
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 3.
5
Ophthalmology Volume -, Number -, Month 2015 involvement in the left eye that was uninvolved 10 years ago at the time of first presentation to us, owing to a marked diagnostic delay that caused multiple recurrences. At the last visit at 8 months after the initial visit, BCVA was 6/6 and 6/12 in the right and left eyes, respectively.
Discussion This is the first study of diagnostic PPV demonstrating the presence of M. tuberculosis DNA in the vitreous fluid of eyes with MSC. In this series, different molecular techniques established a definitive diagnosis of tuberculosis, such as multitargeted PCR analysis in 10 of 11 patients (previously reported sensitivity of 77.8%),10 the MTBDRplus assay in 6 cases, and the Gene Xpert MTB/ RIF assay in 4 cases. Multifocal serpiginous choroiditis presents as an isolated ocular disease, and definitive evidence of tuberculosis is extremely rare. The etiologic diagnosis is dependent on the clinical features, corroborative evidence, and response to ATT.1 Bhuibhar and Biswas21 substantiated a tubercular cause in a patient with amphigenous choroiditis by performing nested PCR analysis of an aqueous sample. Although most patients with MSC respond favorably to empirical treatment with 4-drug ATT and corticosteroids, a few patients fail to respond and pose a significant diagnostic and therapeutic challenge. Drug resistance, paradoxical worsening related to the toxic effects of ATT, poor compliance, or a nontubercular cause may be thought of as possible reasons in these patients. Although an invasive procedure, diagnostic PPV answers some of these questions. Emergence of drug resistance poses a major limitation in effectively controlling tuberculosis-related diseases, pulmonary or extrapulmonary. The conventional methods to detect resistance to rifampicin or isoniazid include culture of the mycobacteria and drug susceptibility testing, which are slow and time consuming. This leads to a delay in initiating appropriate therapy, allowing transmission and spread of MDR tuberculosis, particularly pulmonary tuberculosis. In the case of ocular involvement, this results in treatment failure, relapse, and significant visual morbidity resulting from uncontrolled intraocular inflammation. However, fear of overdiagnosing tuberculosis uveitis in an endemic region may lead to inadvertent use of corticosteroids and immunosuppressive agents. Therefore, in highprevalence countries, rapid diagnosis of tuberculosis is a prerequisite for administering targeted therapy to uveitis patients. Also, detection of MDR tuberculosis in these eyes by identifying resistance to rifampicin or isoniazid, as in other extrapulmonary sites, may save these eyes from blindness.
Significant advances in molecular techniques have offered the ability to obtain an early diagnosis of tuberculosis. In 2010, the World Health Organization approved the Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis.22 In addition to detecting M. tuberculosis DNA, it allows simultaneous testing of susceptibility to rifampicin (rifampicin) in a short time (approximately 2 hours). It is a closed cartridge system, based on a realtime heminested PCR that amplifies the rpoB gene 81-bp rifampicin resistanceedetermining region at codons 507 through 533. Several studies have reported high sensitivities and specificities from pulmonary and extrapulmonary samples.23e27 In our present series, the Gene Xpert assay could detect the M. tuberculosis genome in only 4 of 10 patients with positive PCR results, indicating a low sensitivity. The line probe assay (GenoType MTBDRplus) is another test in the rapid diagnostic algorithm of M. tuberculosis, allowing results within approximately 5 hours. In addition to detecting mutations associated with the rpoB gene for rifampicin resistance, the MTBDRplus assay detects katG and inhA gene mutations for high-level and low-level isoniazid resistance, respectively. It has a higher detection rate for rifampicin resistance compared with isoniazid resistance.28e30 In our study, the MTBDRplus assay detected M. tuberculosis in 6 eyes, of which rifampicin monoresistance was identified in 2 and combined rifampicin plus isoniazid resistance was detected in 1. On comparing the 3 tests for detecting M. tuberculosis in our patients, multitargeted PCR showed positive results in 10 of 11 eyes, the MTBDRplus assay showed positive results in 6 of 11 eyes, and the Gene Xpert assay showed positive results in 4 of 11 eyes. When comparing the performance of the 2 assays (Gene Xpert and MTBDRplus) for detecting MDR tuberculosis, the Gene Xpert assay detected rifampicin resistance in only 1 eye, whereas the MTBDRplus assay detected rifampicin resistance in 4 eyes (including 1 with isoniazid resistance). False-positive or overreporting of rifampicin resistance, which have been associated with the Gene Xpert assay,23 were not found in our study. On the contrary, the only patient detected by Gene Xpert as being resistant to rifampicin in our series was resistant to rifampicin by the MTBDRplus assay as well, as confirmed by rpoB gene sequencing to have a mutation at codon 526 (case 1). We found that although the Gene Xpert is preferable for faster detection of MDR tuberculosis (in approximately 2 hours), the MTBDRplus assay was superior in testing drug susceptibility. It additionally detected rifampicin monoresistance in 1 patient (case 11) who demonstrated positive PCR results but negative Gene Xpert results.
Figure 3. A, Fundus photograph of the right eye of a 21-year-old woman (case 1) with positive tuberculin skin test results showing active lesions of multifocal serpiginous choroiditis in the posterior pole along with healed lesions. B, Fundus photograph (left) and fluorescein angiograms (middle and right) showing progression of lesions 1 week after initial presentation. C and D, Fundus photographs showing relentless progression of lesions at (C) 3 and (D) 5 weeks, despite oral corticosteroids and antitubercular therapy. E, rpoB gene sequencing of vitreous fluid from case 1 showing a mutation at 526 codon (CAC [wild type]dTAC [mutant type]), within an 81-bp region of the rifampicin resistanceedetermining region of the rpoB gene. F and G, Fundus photographs obtained after diagnostic pars plana vitrectomy of the right eye and initiation of antiemultidrug-resistant tuberculosis therapy based on detection of rifampicin resistance; the patient showed marked resolution of lesions at (F) 4 weeks and (G) 8 months.
6
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 4. A, Fundus photographs from a 43-year-old man with positive tuberculin skin test results showing (left) active lesions of multifocal serpiginous choroiditis in the right eye at initial presentation in 2003 and (right) a normal left eye (right). B, Fundus photographs obtained after treatment with antitubercular therapy and corticosteroids. The lesions in the right eye healed and both eyes remained stable until 2007. C, Fundus photographs showing that although the lesions in the right eye remained scarred, fresh lesions developed in the fellow eye in the macula in 2008, for which retreatment with antitubercular therapy, corticosteroids, and immunosuppressive agents was administered. DeG, Fundus photographs of the left eye showing remitting and relapsing lesions during approximately the next 5 years: (D) 27, (E) 53, (F) 58, (G) and 62 months after the first appearance of lesions in the left eye. H, Montage fundus view and fluorescein angiogram of the left eye showing active lesions in the posterior pole as well as the periphery before performing diagnostic pars plana vitrectomy in the left eye. I, rpoB gene sequencing of vitreous fluid from case 11 showing a mutation at 531 codon (TCG [wild type]d TTG [mutant type]) within an 81-bp region of a rifampicin resistanceedetermining region of the rpoB gene. J, Fundus photograph of left eye 7 months after initiating antiemultidrug-resistant tuberculosis therapy showing healed lesions.
Because of an absence of overt systemic tuberculosis and lack of a histopathologic evidence in MSC, the exact pathogenesis remains unknown. The immune-mediated hypersensitivity reaction triggered by tuberculosis antigens has been one possible explanation.9 Although Rao et al9 demonstrated the presence of the M. tuberculosis within the retinal pigment epithelium (RPE) of a patient with panuveitis, our results demonstrate its presence in the
vitreous fluid of patients with active MSC and latent tuberculosis. Further, the role of RPE serving as a reservoir for intraocular M. tuberculosis infection recently was proposed by Nazari et al31 in human fetal RPE cells. The evidence of M. tuberculosis DNA in both of these intraocular sites (vitreous or RPE) reinforces the role of M. tuberculosis in causing or reactivating the inflammation of the inner choroid and RPE in MSC.
7
Ophthalmology Volume -, Number -, Month 2015
Figure 4. (continued).
8
Bansal et al
Diagnostic Vitrectomy in MSC
Figure 4. (continued).
Evidence of tuberculosis in MSC is growing not only from endemic areas, but also from nonendemic areas, particularly in people migrating from endemic areas.4,8,32 We suggest looking for tuberculosis in these patients. Detection of rifampicin resistance in the present series led to revision of diagnosis to MDR tuberculosis and hence the treatment in 3 patients who showed poor response to initial therapy. Diagnostic PPV is associated with a decreased use of immunosuppressive therapy.33 Also, timely detection of rifampicin resistance can prevent inadvertent initiation of immunosuppressive drugs for possible paradoxical worsening of MSC. It emphasizes the need for a high index of suspicion of MDR tuberculosis in patients with MSC showing atypical features or poor response to therapy. Nazari et al31 have suggested the possibility of RPE cells as the site for latent tuberculosis as well as the site for reactivation of tubercular posterior uveitis. Negative results by tuberculosis PCR, Gene Xpert MTB/RIF assay, and MDRTBplus assay in 1 of our patients who also had no evidence of latent tuberculosis (patient 9) suggests the absence of local (ocular) or systemic M. tuberculosis antigenic stimulus in this patient and strengthens the causative association of latent tuberculosis with MSC in an endemic population. Although the beneficial effects of diagnostic PPV are well known in terms of adequate amount of vitreous sample for multiple tests,14,15,33 it has an added advantage over aqueous sampling in eyes with MSC because they usually have minimal or no inflammation in the anterior chamber.1
However, the potential adverse effects of routine vitreous surgery cannot be ignored, and only an experienced vitreoretinal surgeon should perform a diagnostic PPV, especially in eyes with good visual potential. Our study has a few limitations. We did not perform QuantiFERON-TB Gold In-Tube testing for all patients. Although this is a hot topic of discussion, and negative results effectively rule out tuberculosis, a substantial number of patients may have false-positive results.34 The MTBDRplus assay was performed on frozen aliquots, whereas the multitargeted PCR analysis and the Gene Xpert assay were performed on fresh samples. A possible bacterial disintegration could have impaired M. tuberculosis detection by the MTBDRplus assay and reduced the sensitivity of the test. This may have caused a discrepancy in the results. Furthermore, the comparison of laboratory (multitargeted PCR) and nonlaboratory (Gene Xpert and MTBDRplus assay) methods for detecting MDR tuberculosis in this study also is limited by a small sample size. Another limitation is a short follow-up. Therefore, long-term results of response to a targeted (rather than empirical) therapy, particularly MDR tuberculosis treatment, are not known. We conclude that in an endemic region the M. tuberculosis genome could be detected by different molecular techniques in eyes with MSC in patients who also had evidence of latent tuberculosis. A high index of suspicion should be maintained for rifampicin resistance in patients showing atypical features or poor response to initial conventional therapy.
9
Ophthalmology Volume -, Number -, Month 2015
References 1. Bansal R, Gupta A, Gupta V, et al. Tubercular serpiginouslike choroiditis presenting as multifocal serpiginoid choroiditis. Ophthalmology 2012;119:2334–42. 2. Gupta V, Gupta A, Arora S, et al. Presumed tubercular serpiginouslike choroiditis: clinical presentations and management. Ophthalmology 2003;110:1744–9. 3. Bansal R, Gupta A, Gupta V, et al. Role of anti-tubercular therapy in uveitis with latent/manifest tuberculosis. Am J Ophthalmol 2008;146:772–9. 4. Vasconcelos-Santos DV, Rao PK, Davies JB, et al. Clinical features of tubercular serpiginous like choroiditis in contrast to classic serpiginous choroiditis. Arch Ophthalmol 2010;128: 853–8. 5. Schatz H, Maumenee AE, Patz A. Geographic helicoid peripapillary choroidopathy. Trans Am Acad Ophthalmol Otolaryngol 1974;78:747–61. 6. Jabs DA, Rosenbaum JT, Foster CS, et al. Guidelines for the use of immunosuppressive drugs in patients with ocular inflammatory disorders: recommendations of an expert panel [review]. Am J Ophthalmol 2000;130:492–513. 7. Hooper PL, Kaplan HJ. Triple agent immunosuppression in serpiginous choroiditis. Ophthalmology 1991;98:944–51. 8. Khanamiri HN, Rao NA. Serpiginous choroiditis and infectious multifocal serpiginoid choroiditis. Surv Ophthalmol 2013;58:203–32. 9. Rao NA, Saraswathy S, Smith RE. Tuberculous uveitis: distribution of Mycobacterium tuberculosis in the retinal pigment epithelium. Arch Ophthalmol 2006;124:1777–9. 10. Sharma K, Gupta V, Bansal R, et al. Novel multi-targeted polymerase chain reaction for diagnosis of presumed tubercular uveitis. J Ophthalmic Inflamm Infect 2013;3:25. 11. Priya K, Madhavan HN, Reiser BJ, et al. Association of herpes-viruses in the aqueous humor of patients with serpiginous choroiditis: a polymerase chain reaction-based study. Ocul Immunol Inflamm 2002;10:253–61. 12. Mahendradas P, Kamath G, Mahalakshmi B, et al. Serpiginous choroiditis-like picture due to ocular toxoplasmosis. Ocul Immunol Inflamm 2007;15:127–30. 13. Sharma K, Bansal R, Sharma A, et al. Successful treatment of rifampicin resistant intraocular tuberculosis. Ocul Immunol Inflamm 2014 Mar 21. [Epub ahead of print]. 14. Mruthyunjaya P, Jumper JM, McCallum R, et al. Diagnostic yield of vitrectomy in eyes with suspected posterior segment infection or malignancy. Ophthalmology 2002;109:1123–9. 15. Davis JL, Miller DM, Ruiz P. Diagnostic testing of vitrectomy specimens. Am J Ophthalmol 2005;140:822–9. 16. American Thoracic Society. Diagnostic standards and classification of tuberculosis and other mycobacterial diseases (14th ed). Am Rev Respir Dis 1981;123:343–58. 17. Taniguchi H, Aramaki H, Nikaido Y, et al. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol Lett 1996;144:103–8. 18. Read SJ, Kurtz JB. Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay. J Clin Microbiol 1999;37: 1352–5.
10
19. Zhang Y, Kimura T, Fujiki K, et al. Multiplex polymerase chain reaction for detection of herpes simplex virus type 1, type 2, cytomegalovirus, and varicellazoster virus in ocular viral infections. Jpn J Ophthalmol 2003;47:260–4. 20. Mahalakshmi B, Therese KL, Shyamala G, et al. Toxoplasma gondii detection by nested polymerase chain reaction in lens aspirate and peripheral blood leukocyte in congenital cataract patients: the first report from a tertiary eye hospital in India. Curr Eye Res 2007;32:653–7. 21. Bhuibhar SS, Biswas J. Nested PCR-positive tubercular ampiginous choroiditis: a case report. Ocul Immunol Inflamm 2012;20:303–5. 22. World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system. Policy statement. Geneva, Switzerland: World Health Organization; 2011. Available at: http://apps.who.int/iris/ handle/10665/44586. Accessed on 22nd July, 2014. 23. Boehme CC, Nabeta P, Hillemann D, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010;363:1005–15. 24. Marlowke EM, Novak Weekley SM, Cumpio J, et al. Evaluation of the Cepheid Xpert MTB/RIF assay for the direct detection of Mycobacterium tuberculosis complex from respiratory specimens. J Clin Microbiol 2011;49:1621–3. 25. Hillemann D, Ruesch-Gerdes S, Boehme C, Richter E. Rapid molecular detection of extrapulmonary tuberculosis by automated GeneXpert MTB/RIF system. J Clin Microbiol 2011;49:1202–5. 26. Nhu NTQ, Heemskerk D, Thu DDA, et al. Evaluation of GeneXpert MTB/RIF for diagnosis of tuberculous meningitis. J Clin Microbiol 2014;52:226–33. 27. Lawn SD, Nicol MP. Xpert MTB/RIF assay: development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin resistance. Future Microbiol 2011;6:1067–82. 28. Ling D, Zwerling A, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta-analysis. Eur Respir J 2008;32:1165–74. 29. Albert H, Bwanga F, Mukkada S, et al. Rapid screening of MDR-TB using molecular line probe assay is feasible in Uganda. BMC Infect Dis 2010;10:41. 30. Farooqi JQ, Khan E, Alam SM, et al. Line probe assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan. J Pak Med Assoc 2012;62:767–72. 31. Nazari H, Karakousis PC, Rao NA. Replication of Mycobacterium tuberculosis in retinal pigment epithelium. JAMA Ophthalmol 2014;132:724–9. 32. Gan WL, Jones NP. Serpiginous-like choroiditis as a marker for tuberculosis in a non-endemic area. Br J Ophthalmol 2013;97:644–7. 33. Oahalou A, Schellekens PAWJF, De Groot-Mijnes JD, Rothova A. Diagnostic pars plana vitrectomy and aqueous analyses in patients with uveitis of unknown cause. Retina 2014;34:108–14. 34. Pepple KL, VanGelder R, Forooghian F. Caveats about QuantiFERON-TB Gold In-Tube testing for uveitis. Am J Ophthalmol 2014;157:752–3.
Bansal et al
Diagnostic Vitrectomy in MSC
Footnotes and Financial Disclosures Originally received: August 27, 2014. Final revision: November 18, 2014. Accepted: November 18, 2014. Available online: ---.
*Both Dr. Bansal and Dr. Sharma contributed equally as first authors.
Manuscript no. 2014-1384.
1
Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India. 2 Department of Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India. 3
Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
4
Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
5
Bureau of Health Laboratories, Jacksonville, Florida.
Presented as a poster at: American Academy of Ophthalmology Annual Meeting, October 2014, Chicago, Illinois.
Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, India. Abbreviations and Acronyms: ATT ¼ antitubercular therapy; BCVA ¼ best-corrected visual acuity; MDR ¼ multidrug-resistant; MSC ¼ multifocal serpiginoid choroiditis; PCR ¼ polymerase chain reaction; PPV ¼ pars plana vitrectomy; RPE ¼ retinal pigment epithelium; TST ¼ tuberculin skin test. Correspondence: Amod Gupta, MS, Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India 160012. E-mail: [email protected].
11