Accepted Manuscript Title: Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes Authors: Shrikala Baliga, Christina Murphy, Leesha Sharon, Suchitra Shenoy, Dhanashree Biranthabail, Helena Weltman, Steve Miller, Ranjan Ramasamy, Jyotsna Shah PII: DOI: Reference:
S1201-9712(18)34469-2 https://doi.org/10.1016/j.ijid.2018.07.011 IJID 3288
To appear in:
International Journal of Infectious Diseases
Received date: Revised date: Accepted date:
18-3-2018 7-7-2018 9-7-2018
Please cite this article as: Baliga Shrikala, Murphy Christina, Sharon Leesha, Shenoy Suchitra, Biranthabail Dhanashree, Weltman Helena, Miller Steve, Ramasamy Ranjan, Shah Jyotsna.Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes.International Journal of Infectious Diseases (2018), https://doi.org/10.1016/j.ijid.2018.07.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA
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probes
Shrikala Baliga 1, Christina Murphy 2, Leesha Sharon 1, Suchitra Shenoy 1, Dhanashree
Biranthabail 1, Helena Weltman 2, Steve Miller 3, Ranjan Ramasamy 2 and Jyotsna Shah 2* 1
Kasturba Medical College, Manipal Academy of Higher Education, Mangaluru, Karnataka,
India 2
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ID-FISH Technology, Palo Alto, CA, USA
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University of California, San Francisco, CA, USA
* Corresponding author: Shah J, ID-FISH Technology Inc., 797 San Antonio Road, Palo Alto,
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CA 94303. Telephone: 1-650-543-2641, Email:
[email protected]
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Orcid ID:
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Jyotsna Shah: 0000-0001-9890-366X
Ranjan Ramasamy: 0000-0003-0246-7053
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Shrikala Baliga: 0000-0002-6722-1532 Suchitra Shenoy: 0000-0003-1425-0097
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Dhanashree Biranthabail: 0000-0002-5370-0663
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Running title: FISH sputum test for TB
Note: Preliminary results from this study were presented as a poster at the 17th International Congress of Infectious Diseases, Hyderabad, India in 2016 with poster abstract published in the International Journal of Infectious Diseases 45S, page 411 (2016) https://doi.org/10.1016/j.ijid.2016.02.878.
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Highlights
Rapid and inexpensive identification and differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria in sputum needed Genus and species-specific dual colour FISH assay meets need Requires only a light microscope with LED/filter unit Simple and suitable for resource-limited laboratories
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Abstract
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Purpose: Mycobacterium tuberculosis in sputum is mainly detected in resource-limited
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tuberculosis-endemic countries by acid-fast bacilli (AFB) staining and identifying sputum-derived
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cultures. PCR techniques are practical only in well-resourced laboratories. We investigated the
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application of a rapid, simple and inexpensive fluorescence in situ hybridization (FISH) assay to identify and differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria
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(NTM) in sputa.
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Methods: The Mycobacterium/Nocardia Genus (MN Genus)-MTBC FISH assay utilized two different DNA probes labelled with different fluorescent molecules that hybridize respectively with 16S rRNA of the genus Mycobacterium and 23S rRNA of MTBC. The assay was tested on 202 patient sputum samples in Mangaluru, Karnataka state, India. Sputa were first liquefied and
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bacteria concentrated before performing the FISH assay and parallel culturing and staining for acid fast bacilli (AFB). Identities of cultured bacteria from DNA sequencing were compared with FISH assay findings on corresponding sputa.
Results: Of the 202 sputum samples, 67 reacted with both MN Genus-specific and MTBC-
specific probes, none reacted only with the MTBC-specific probe and 22 reacted only with the MN Genus-specific probe. The FISH assay yielded results in two hours and had a limit of
detection of 2.2 x 104 cfu per ml in sputum spiked with cultured M. tuberculosis. The diagnostic
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sensitivity, specificity, and positive and negative predictive values of the FISH assay were 89.7%, 95.5%, 88.0% and 92.6% respectively for MTBC in patient sputa. NTM were a
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significant cause of tuberculosis-like infections in Mangaluru.
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Conclusions: The MN Genus-MTBC dual probe fluorescence FISH assay previously applied to cultures can also be utilized in resource-limited tuberculosis-endemic countries for rapidly
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identifying and differentiating MTBC and NTM in sputum samples.
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Key Words: Fluorescence in situ hybridization, LED fluorescence microscopy, Mycobacterium
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tuberculosis, non-tuberculous mycobacteria, tuberculosis diagnosis, sputum assay
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INTRODUCTION
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Infection with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, is estimated to have caused 1.67 million deaths worldwide in 2016 with 26% of these occurring in India [1]. Most human pulmonary mycobacterial infections are caused by MTB and closely
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related species such as Mycobacterium bovis that together comprise the Mycobacterium tuberculosis complex (MTBC) but clinically relevant infections with non-tuberculous mycobacteria (NTM) also occur worldwide [2, 3]. The management and treatment of patients with MTBC tuberculosis and NTM infections is different, particularly because many NTM are resistant to common drugs used to treat MTBC infections, thus making their differential diagnosis important. Human NTM infections are caused mainly by species of the
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Mycobacterium avium complex (MAC) and less frequently by other Mycobacteria such as M. kansasii, M. fortuitum, M. xenopi, M. abscessus, and M. simiae [4 – 8]. Nocardia species, that are common in the environment and closely related to mycobacteria, can at times cause
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tuberculosis-like disease [9].
Conventional detection of MTB in tuberculosis-endemic countries is through microscopic examination for acid-fast staining bacilli (AFB), usually by Ziehl-Neelsen (ZN) staining, in
smears of respiratory samples, most commonly sputum, [10] and subsequently identifying mycobacteria cultured from such samples. However, AFB staining methods do not easily
differentiate between Mycobacterium species and the closely related Nocardia species which may also be found in respiratory samples. The correct identification of mycobacteria from
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sputum-derived cultures at the species level in resource-limited tuberculosis-endemic relies on observing colony characteristics and additional biochemical, immunological or molecular
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biological tests [11-13]. Culture-based identification however requires good quality sputum
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samples, and is time consuming because it needs 2-6 weeks to produce results and is labour
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intensive.
Several different tests based on nucleic acid amplification (NAA) have been developed
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to identify MTB in sputum samples but they vary considerably in their sensitivity and specificity
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[14]. The Xpert MTB/RIF system (Cepheid, Sunnyvale, CA, USA), a test based on PCR amplification and detection of relevant DNA sequences in MTB, is more sensitive than AFB microscopy and commonly used as the initial diagnostic test for MTB and rifampicin resistance
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in the sputum of patients in clinical laboratories that possess the necessary equipment and expertise [15]. Two other NAA tests have been approved by the Food and Drugs Administration
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of the USA (FDA) for use with sputum samples [16]. These are the Amplified Mycobacterium Tuberculosis Direct Test (Gen-Probe, San Diego, CA) and the Amplicor Mycobacterium Tuberculosis Test (Roche Diagnostic Systems, Branchburg, NJ). The FDA-approved NAA tests
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presently identify only MTB and provide results in 24-48h, but cost and infrastructure requirements limit their use in resource-limited countries. There is now consensus on the need for a NAA assay to detect MTB that is capable of yielding results from respiratory samples in 2h or less [17]. The suitability of such a test for use in resource-limited countries would be an additional advantage. However, NAA-based tests are prone to inhibition by a variety of
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components present in biological samples, such as sputum, that reduce their sensitivity and require the use of elaborate controls [16, 17].
Several fluorescence in situ hybridization (FISH) assays that target multiple copies of
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rRNA present in mycobacterial cells, and therefore do not require NAA, have been described [18 - 25]. Such FISH assays are based on DNA [18 - 21] or peptide nucleic acid [22 - 25] probes but are not yet in widespread clinical use. Because NAA is not necessary, FISH assays are not affected by inhibitors of NAA, and do not require the expensive equipment and infrastructure
needed for NAA. They are therefore more suitable for resource-limited tuberculosis-endemic countries. With two exceptions [18, 24], the FISH assays reported for MTB and other
mycobacteria have largely been applied to cultures and biopsied tissues. The application of
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FISH assays to sputum samples is more complex than for cultures because of a lower MTB concentration, the presence other bacteria in sputum and a need to liquefy sputum before
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preparing smears for the assays. A DNA probe- based FISH assay has been used to identify
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MTB in a large number of sputum samples with a reported sensitivity and specificity of 82% and 98% respectively compared with traditional culture-based identification, but this FISH assay did
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not differentially identify MTB and NTM [18]. A PNA-based FISH assay differentiated MTB from several NTM species but had relatively low sensitivity with sputum samples [24]. We described
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two dual fluorescence FISH assays viz. the MN Genus-MTBC and the MAC-MTBC assays,
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which can identify MTBC and MAC with specificity and sensitivity of 100% in a large number of cultures and also differentiate them from other NTM and Nocardia species [19]. We report here on a method for applying the MN Genus-MTBC FISH assay to identify MTBC and differentiate it
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from NTM in sputum samples and the relevant clinical diagnostic parameters. The study aimed to meet an important diagnostic need in resource-limited countries with a high burden of
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tuberculosis.
MATERIALS AND METHODS
Sputum Samples and Processing for FISH, AFB Staining and Culture
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A total of 202 left over, decoded sputum samples from 143 patients suspected to have tuberculosis by clinical criteria were collected from hospitals in Mangaluru, India for use in the study. They were composed of two samples each collected at separate times from 59 patients and one sample each collected from 84 other patients. The sputum samples were processed by
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a previously described method for liquefying sputum and concentrating bacilli to prepare smears for acid-fast staining [26, 27]. In summary, sputum samples were mixed with sputum liquefying reagent containing guanidinium hydrochloride, sarkosyl and Tris (2-carboxyethyl) phosphine hydrochloride, pH 7.2, by inversion 10-15 times and then incubated for 10min at ambient
temperature with intermittent mixing. The samples were then syringe filtered with glass wool and centrifuged at 3000g for 10min. The pellet containing concentrated bacteria was used to make smears on glass microscope slides that were then fixed with 5% phenol in 70% ethanol for
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5min. Duplicate air-dried smears were used for FISH assays and ZN staining for AFB. BSL3
laboratory safety guidelines were followed throughout when handling sputum samples. Fixation
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in 5% v/v phenol in 70% v/v ethanol renders the fixed smears non-infectious and safe for
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handling outside a biosafety facility [28]. After preparing smears for FISH and ZN staining, remains of the concentrated pellets were subsequently treated with sodium hydroxide, pH
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Mycobacterial Cultures
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neutralized and used as an inoculum for both liquid and solid cultures.
Liquid cultures were grown at Kasturba Medical College, Mangaluru, India by inoculating TM
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the processed pellets containing bacteria from sputum samples into MGIT tubes in the BACTEC MGIT TM 320 detection system (Becton Dickinson, Franklin Lakes, NJ) as previously
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described [19]. The processed pellets from sputum samples were also used to initiate cultures on Lowenstein Jensen (LJ) solid media slants (BD Diagnostics, Sparks, MD) [19]. Mycobacteria
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grown in liquid and solid cultures were identified at the species level by DNA sequencing.
Sequencing of 23S rDNA From Sputum-Derived Mycobacterial Cultures
DNA was purified with the Generation Capture Column kit (Qiagen, Valencia, CA) from microscope slide smears made from cultures and then sequenced in the ID-FISH laboratories, Palo Alto, CA as previously described [19]. Briefly, the relevant 23S rDNA region was amplified
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by PCR using the forward Myco-F and reverse Myco-R primers [19]. The approximately 200bp PCR products were subsequently sequenced using the same Myco-F and Myco-R primers. The resulting sequences were compared by BLAST analysis with published 23S rDNA sequences for identifying species of mycobacteria. A sputum sample was considered to contain MTBC if
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either one or both of the derived solid or liquid cultures yielded mycobacteria that could be identified as MTBC by DNA sequencing. FISH Assay Reagents
The Mycobacterium/Nocardia Genus (MN Genus) and MTBC combination FISH assay
reagents were provided by ID-FISH Technology Inc., Palo Alto, CA as a kit (MN Genus-MTBC
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FISH, catalogue number MycoGTK04) and used on sputum pellet smears by the method described previously for identifying cultures [19]. The kit contained two DNA probes,
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pretreatment buffer, pretreatment rinse buffer, hybridization solution, wash buffer, and mounting
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medium [29 - 31]. One probe was a MN Genus-specific probe complementary to an internal 16S rRNA sequence shared between the Mycobacterium and Nocardia genera [19, 29]. This probe
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recognized 25 of 26 different NTM species, 4 of 4 different MTBC species, 3 of 5 different Corynebacterium species and 3 of 3 different Nocardia species but not 22 other common
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human bacterial and protozoan pathogens when tested on cultured organisms [19]. BLAST
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analysis against published 16S rRNA sequences in the National Centre for Biological Information (NCBI) database showed that significant sequence homology was only seen with members of the Mycobacteriaceae, Nocardiaceae and Corynebacteriaceae families, which are
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closely related families belonging to the order Actinomycetales. The other probe was a MTBCspecific probe that was complementary to a 23S rRNA 5’ sequence unique to the MTBC species
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complex [19, 29, 30]. BLAST analysis against 23S rRNA sequences in the NCBI database showed that MTBC-specific probe sequence was identical only with 23S rRNA of MTBC organisms. Furthermore, the MTBC-specific probe only hybridized to MTBC complex species
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and not 26 different NTM species or 22 other common human bacterial and protozoan pathogens when tested on cultured organisms [19]. The MN Genus-specific DNA probe was conjugated with a green fluorescent dye Atto 480 and the MTBC-specific DNA probe conjugated with an orange fluorescent dye Atto 550 to permit dual color fluorescence-based identification of mycobacteria [19].
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FISH Assay Procedure
MN Genus-MTBC dual color fluorescence FISH assay on slide smears was performed at Kasturba Medical College, Mangaluru, India as previously described [19]. Selected smears were
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also independently tested by the FISH assay at the ID-FISH laboratories, Palo Alto, CA. In summary, after the addition of 1 ml of a proprietary pre-treatment buffer to each fixed smear to permeabilise cells, the slides were placed in a humid chamber at 37°C for 15min. The slides
were then removed from the incubator, rinsed with pre-treatment rinse buffer and air-dried. After the subsequent addition of 10 µl of appropriate chaotropic hybridization solution (guanidinium
salts and the non-ionic detergent IGEPAL) and two DNA probes, each smear was covered with a plastic cover-slip and placed in a 37°C humid chamber for 15min for hybridization. After
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hybridization, each smear was washed three times for 2min each with wash buffer (saline-
sodium citrate containing sodium dodecyl sulphate) at ambient temperature and then dried in
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complete darkness. A drop of mounting medium was then added and the smear was covered
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with a glass cover-slip. Smears were read at 1000x magnification using a LED light source with custom filter sets (Fraen Corp, Cusago, Italy) for viewing green (excitation 490 nm; emission
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529-530nm band pass filter) and orange fluorescence (excitation 530 nm; emission 575 nm long pass filter) in an Olympus BX laboratory light microscope [19]. In the two dual color
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fluorescence MN Genus-MTBC FISH assay performed on a single slide smear as previously
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shown for cultures, all Mycobacterium and Nocardia species and some Corynebacterium species fluoresce green with the MN-Genus probe, while only MTBC cells fluoresce orange with
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the MTBC-specific probe [19].
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Determination of the Limit of Detection of MTBC in Sputum in the FISH Assay With an approximate starting concentration of 106 to 107 cells per ml from MGIT cultures,
serial ten-fold dilutions in PBS up to a dilution of 10−7 were prepared from three replicate MTB
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(ATCC 25177) cultures. At each dilution, 10 μl samples were plated on Middlebrook 7H10 agar plates and incubated at 370C, 5% v/v CO2 for up to six weeks. Colonies were counted after sufficient growth was obtained to determine the number of colony forming units (cfu) at each dilution. An aliquot of 200 µl of each dilution of MTB was added to 1.8 ml of Mycobacteriumnegative pooled sputum provided by the Mycobacteriology Laboratory, University of California, San Francisco, CA. Sputum samples containing MTB at different concentrations were then
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processed as described above for clinical samples and the pellet of MTB tested in the FISH assay. The limit of detection (LOD) was defined as the arithmetic mean of the lowest concentration of bacteria at which the three smears tested gave a positive reaction in the MN
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Genus-MTBC FISH assay.
Data Analysis
The diagnostic sensitivity, specificity, positive predictive value (PPV) and negative
predictive value (NPV) and their 95% confidence intervals were determined using an online
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statistical calculator [32].
FISH Assays on Sputum Samples
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RESULTS
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Figure 1 provides illustrative results obtained with the MN Genus-MTBC FISH assay
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performed on two different sputum smears that yielded bacterial cultures that were confirmed by DNA-sequencing to be MTB and the NTM Mycobacterium abscessus respectively. The FISH
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assays are readily readable. Clumps of sputum material that may be present tend to weakly autofluoresce, but this does not typically interfere with identifying the characteristic fluorescence
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in FISH-positive mycobacteria.
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(Insert Figure 1 here)
Of the 202 sputum samples tested by FISH, 67 reacted with both MN Genus-specific
and MTBC-specific probes in the MN Genus-MTBC FISH assay, while no sample reacted only with the MTBC-specific probe. However, 22 of the 202 samples reacted only with the MN Genus- specific probe and not the MTBC-specific probe. Of these 22 samples, eight were ZNAFB positive samples. This included five of the six samples that were confirmed as NTM by DNA sequencing of resulting cultures and three that could not be sequenced. The six culture
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positive samples, confirmed as mycobacteria by DNA sequencing, included two samples of Mycobacterium abscessus, one Mycobacterium avium, one Mycobacterium immunogenum, one Mycobacterium pheli and one Mycobacterium smegmatis. Taking cultures that could be identified by DNA sequencing as the standard, the results suggest that the MN Genus probe
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has a diagnostic sensitivity and specificity of 100% in sputum samples.
The results of the MTBC-specific FISH staining reaction on all 202 sputum samples in
relation to DNA sequence identification of MTBC in the corresponding sputum-derived cultures and sputum ZN-AFB staining and are summarized in Tables 1 and 2 respectively. The
identification of MTBC-specific 23S rDNA sequence in either liquid or solid culture was regarded
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as showing the definitive presence of MTBC in the original sputum sample.
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(Insert Tables 1 & 2 here)
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Of the 202 sputum samples analyzed with the results being shown in Tables 1 and 2, 118 samples were from 59 patients who had each provided two samples of sputum at different
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times. The results from these 59 patients were analyzed separately and are shown in Table 3. An MTBC FISH on either of the two sputum samples and correspondingly MTBC sequence
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identification in either of the two derived cultures, was considered to be a positive demonstration
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of MTBC by the respective techniques. Analysis of results from the 59 patients showed that the diagnostic sensitivity, specificity, PPV and NPV of the MTBC FISH assay were not significantly
and 3).
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different from the those obtained by considering the 202 sputum samples individually (Tables 2
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(Insert Table 3 here)
The results (Tables 2 and 3) also show that the sensitivity of the MTBC FISH assay
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tends to be greater with sputum samples that are ZN-AFB positive than those which are not, and that this difference tends to be more marked when two sputum samples from a patient are examined (Table 3). Limit of Detection of MTBC in Sputum by the FISH Assay
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The LOD with the MTBC-specific probe on the three replicate MTB samples were 1.6 x 104, 1.7 x 104 and 3.2 x 104 cfu per ml giving an average LOD in sputum of 2.2 x 104 cfu per ml. The same LOD was obtained with the MN Genus-specific probe in the dual fluorescence MN
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Genus-MTBC FISH assay.
DISCUSSION
The results show that the MN Genus-MTBC dual probe fluorescence FISH assay,
previously used for detecting MTBC in cultures [19], can be also applied for detecting MTBC on appropriately processed sputum samples. The diagnostic sensitivity and specificity of the MN
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Genus probe of 100% with sputum samples is the same as that previously observed with
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cultured bacteria [19]. When used to test cultures, the MN Genus-specific probe in the MN Genus-MTBC FISH assay previously detected all common species of Mycobacterium isolated
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from patients as well as all Nocardia species tested and some Corynebacterium species [19].
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Corynebacterium, Nocardia and Mycobacterium are related genera belonging to the order Actinomycetales. Nocardia species are partially ZN-AFB stain positive, while Corynebacterium species are not AFB positive. Therefore, the MN Genus-MTBC FISH assay, used in
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conjunction with ZN-AFB staining, can identify sputum as containing MTBC or NTM/Nocardia.
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AFB staining alone does not achieve this. The differentiation of NTM and Nocardia in sputum samples that react only with the MN Genus-specific probe needs to be achieved through
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culture-based identification. While the Xpert MTB/RIF system specifically identifies MTB, it does not show the presence of NTM or Nocardia. If the previously described complementary MTBCMAC FISH assay [19] is simultaneously performed on sputum samples, the non-MTBC
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mycobacteria identified in the MN Genus-MTBC assay can potentially be classified as MAC or other NTM/Nocardia. Mixed infections of MTBC and NTM/Nocardia can also be detected as
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shown possible for cultures [19] if sufficient numbers of the two types of bacteria are present in sputum. In high burden countries like India [19] and China [2, 18] where NTM are a significant cause of pulmonary disease resembling tuberculosis, distinction between MTBC and NTM/Nocardia is important for patient management and treatment. The MN Genus-MTBC FISH assay detects viable bacilli in sputum because RNA is rapidly degraded on cell death. It additionally reveals bacterial morphology. The potential application of the MN Genus-MTBC FISH assay to bronchial washings and tissue biopsies also merits further investigation.
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The LOD for MTB in sputum with the MN Genus-MTBC FISH assay of 2.2 x 104 cfu per ml is comparable to the LOD for ZN or auramine-rhodamine fluorescence staining for AFB [33], and consistent with the FISH assay and ZN staining results from clinical sputum samples
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reported here. However, this LOD was determined by adding MTB from cultures to uninfected sputum. Mycobacteria grow and proliferate optimally in culture and can therefore possess
higher numbers of ribosomes than mycobacteria in sputum samples from patients. Hence the LOD of the MN Genus-MTBC FISH assay in clinical sputum samples may be higher than the LOD obtained by adding cultured MTB to sputum. The LOD for MTB in sputum with the MN
Genus-MTBC FISH assay is however significantly higher than the LOD of
