Rif and multiplex PCR for the diagnosis of tubercular lymphadenitis in HIV-infected patients of North India

Journal Pre-proof Comparative evaluation of loop-mediated isothermal amplification (LAMP) assay, GeneXpert MTB/Rif and multiplex PCR for the diagnosis of tubercular lymphadenitis in HIV-infected patients of North India N. Baikunje, D. Behera, A. Rajwanshi, M. Sharma, A. Sharma, K. Sharma PII:

S0890-8508(19)30308-1

DOI:

https://doi.org/10.1016/j.mcp.2019.101459

Reference:

YMCPR 101459

To appear in:

Molecular and Cellular Probes

Received Date: 19 August 2019 Revised Date:

11 September 2019

Accepted Date: 20 September 2019

Please cite this article as: Baikunje N, Behera D, Rajwanshi A, Sharma M, Sharma A, Sharma K, Comparative evaluation of loop-mediated isothermal amplification (LAMP) assay, GeneXpert MTB/Rif and multiplex PCR for the diagnosis of tubercular lymphadenitis in HIV-infected patients of North India, Molecular and Cellular Probes (2019), doi: https://doi.org/10.1016/j.mcp.2019.101459. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

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MANUSCRIPT

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Title: Comparative evaluation of loop-mediated isothermal amplification (LAMP) assay,

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GeneXpert MTB/Rif and Multiplex PCR for the diagnosis of tubercular lymphadenitis in

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HIV-infected patients of North India.

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ABSTRACT

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Background: Tubercular lymphadenitis (TBLA) is one of the most common extrapulmonary

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manifestations of tuberculosis in patients with HIV. With several other pathological conditions

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presenting as lymphadenitis and lack of consensus regarding a gold standard test, the diagnosis

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of TBLA remains a challenge for the clinician.

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Objectives and design: In this study, we have assessed the potential of loop-mediated

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isothermal amplification (LAMP) test for the diagnosis of TBLA in HIV-infected patients. The

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study group included samples collected by fine needle aspiration (FNAC) of lymph nodes from

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24 HIV-infected patients with TBLA. A composite reference standard was used to identify cases

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of TBLA based on clinical suspicion, results of cytology, AFB smear, MGIT culture, GeneXpert

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MTB/RIF, multiplex polymerase chain reaction (MPCR) and subsequently clinical response to

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antitubercular therapy. These tests were also carried out in 26 control samples of lymph node

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FNAC from HIV-infected patients with non-tubercular lymphadenitis.

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Results: LAMP assay was positive in 19/24 TBLA cases and yielded a sensitivity of 79.17%

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with 100% specificity. Cytology was suggestive in 18/24 (75%) TBLA cases. GeneXpert

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MTB/RIF assay correctly identified 16/24 TBLA cases, but the test did show one false positive

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result reducing its specificity. MPCR had the highest sensitivity of 91.67% as it correctly

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identified 22/24 cases and showed no false positive result.

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Conclusion: The current study highlights the potential of LAMP test for the specific diagnosis of

26

tubercular lymphadenitis in FNAC samples from HIV-infected patients, especially when

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cytology is either non-conclusive or non-available. Though MPCR had a higher sensitivity than

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LAMP assay, the added advantages of low cost, minimal technical expertise and simplicity of

29

procedure make LAMP assay a suitable diagnostic test in resource-limited settings.

30 31 32 33 34 35 36 37

KEYWORDS: M. tuberculosis; nucleic acid amplification test; IS6110; MPB64

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TEXT

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INTRODUCTION

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Out of the many opportunistic infections, tuberculosis is one of the main causes of morbidity and

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death in HIV-infected patients (1, 2). Extra pulmonary tuberculosis is reported more frequently

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and tubercular lymphadenitis (TBLA) accounts for around 50% of extrapulmonary tubercular

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manifestations in HIV-infected patients.(3) TBLA offers a great diagnostic challenge. The

44

clinical presentation overlaps with several other pathological conditions and the laboratory

45

findings are inconsistent in majority of the cases (4). The laboratory diagnosis is usually

46

achieved by cytopathology, presence of acid fast bacilli (AFB) in smear, and culture of

47

mycobacteria from biopsy or more non-invasive fine needle aspiration cytology (FNAC)

48

specimens. These diagnostic methods have advantages and disadvantages of their own and

49

exhibit considerable variation in specificity and sensitivity (5). In cytopathological analysis, the

50

occurrence of epithelioid cell granulomas with or without caseous necrosis is considered as a

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diagnostic marker of tuberculosis. However, cytopathological pattern is not specific for

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tuberculosis and can be shared by other conditions like histoplasmosis, sarcoidosis,

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actinomycosis and vasculitis, making the diagnosis complicated.(6) Although AFB smear using

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Ziehl-Neelsen (ZN) stain is easy and fast, it exhibits a lesser sensitivity missing diagnosis in

55

several cases(7). Further, while fluorescence-based mycobacterium growth indicator tube

56

(MGIT) culture is superior to conventional culture in terms of yield of detection and rapidity, it

57

takes a considerable long time of up to 42 days(8).

58

Recently, nucleic acid amplification techniques (NAAT) notably multiplex polymerase chain

59

reaction (MPCR), which includes simultaneous amplification of several target genes, is being

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used for the diagnosis of TBLA owing to its high sensitivity and rapidity(5). MPCR had a

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sensitivity of 91.3% (smear positive samples) and 86.6% (smear negative samples) for the

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diagnosis of TBLA with a specificity of 100%(9). The commercially-available GeneXpert

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MTB/RIF (GX) assay, a cartridge-based automated test to identify the DNA of Mycobacterium

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tuberculosis, had a sensitivity and specificity of 96.7% and 88.9%, respectively for the diagnosis

65

of TBLA(10, 11).The loop-mediated isothermal amplification (LAMP) assay, a simple molecular

66

test requiring no sophisticated instruments for amplification of DNA(12), could successfully

67

diagnose TBLA from an ulcerated surface lesion(13) and has a reported sensitivity and

68

specificity of 90% and 100% respectively in diagnosing TBLA(14). To the best of our

69

knowledge, there is no literature available on the comparison of these three molecular techniques

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with the conventional technique of cytopathology for the diagnosis of TBLA in HIV-infected

71

patients. This need is indispensable in places where the prevalence of HIV/TB co-infection is

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especially high.

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This study was, therefore, undertaken to assess the diagnostic potential of LAMP assay for

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detecting TBLA in HIV-infected patients and to compare it with cytopathology as well as other

75

molecular techniques of MPCR and GX.

76 77 78

MATERIALS AND METHODS:

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Study design: This study was carried out after approval by Institute Ethics Committee (IEC).

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HIV-infected patients presenting with lymphadenitis to our tertiary care center between July

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2015 to September 2016 were prospectively enrolled for the study. HIV-infected patients with

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already confirmed diagnosis of TB or those who were empirically started on anti-tubercular

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therapy (ATT) were excluded from the study. A written consent was obtained from all the

84

enrolled participants and their clinical profile was registered. One FNAC sample was collected

85

per patient and was coded for blind assessment. One half of that FNAC sample was sent for

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cytological examination by an expert pathologist. Figure 1 shows the typical staining pattern of a

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case of TBLA. The other half was processed according to standard microbiological procedures

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and was subjected to ZN smear, MGIT culture (BD BACTEC MGIT™ 960), molecular analysis

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and a part of the sample was stored at -20oC for future reference. Of about 200µl of sample kept

90

for molecular analysis, 500µl was used for GX while the rest of the sample was subjected to

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DNA extraction for LAMP assay and MPCR.

92

Based on the clinical features and microbiological and cytological findings, the HIV-infected

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study population (n=50) was divided into the following groups, as described previously (12):

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Group I – Confirmed TBLA (n=18); those having culture/smear positive for M. tuberculosis OR

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characteristic epithelioid cell granuloma with caseation AND response to ATT.

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Group II – Suspected TBLA (n=6); those having culture/smear negative for M. tuberculosis OR

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epithelioid cell granuloma on cytology AND response to ATT, in accordance with composite

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reference standard (CRS) described previously. (14)

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Group III – Controls (n=26); HIV-infected patients with lymphadenopathy for reasons other than

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tuberculosis.

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For the purpose of analyzing performance of LAMP, GX and MPCR in diagnosing TBLA in our

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HIV-infected patient population, the patients were grouped as TBLA cases (n=24, confirmed +

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suspected) and non-TBLA cases (n=26, controls).

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For all culture-positive cases, liquid drug susceptibility testing (DST) was carried out for

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streptomycin, isoniazid, rifampicin and ethambutol using MGIT.

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DNA extraction:

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Extraction of DNA was performed using phenol-chloroform-isoamyl alcohol method(15).

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MPCR:

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MPCR was performed using two target genes of M. tuberculosis complex; IS6110 and MPB64

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giving a band

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tuberculosis H37Rv strain was used as positive control and molecular grade water was used as

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negative control.

113

GeneXpert MTB/Rif assay:

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GX (Cepheid, Sunnyvale, CA, USA) testing was performed according to the manufacturer’s

115

instructions. In brief, the sample reagent was added to the sample in a ratio of 2:1. Further the

116

samples were vortexed for 30 s and then made to stand for 15 min at room temperature. 2 ml of

117

the inactivated material was then shifted to the test cartridge and placed in the test platform.

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After an automated cycle lasting about two hours, computed results were used for

119

comparison(17).

120

LAMP:

121

LAMP test was performed targeting the IS6110 gene using three primer pairs, as described

122

previously (12). The amplification was carried out in a water bath set at 63 °C for 45 min

123

followed by termination of reaction at 84 °C for 5 min. H37Rv strain and molecular grade water

124

were simultaneously processed with each run as positive and negative controls, respectively. The

125

amplification was carried out in a water bath. The results were interpreted as positive based on

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appearance of ladder-like pattern on 2% agarose gel electrophoresis or observation of green

127

fluorescence after addition of SYBR green dye(14).

128

Statistical Analysis:

size of 123 bp and 240 bp, as described previously(16). The DNA of M.

129

Standard formulae were used to calculate sensitivity, specificity, positive and negative predictive

130

values.

131 132 133

RESULTS 1. Patient profile:

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The median age of patients was 33 years for TBLA cases and 35 years for controls. 22/24

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(92%) cases and 20/26 (77%) controls were males. The mean duration since the diagnosis of

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HIV was 22 months in cases and 43.34 months in controls (p=0.25). The latest mean CD4 count

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was 347.6 cells/ml among cases and 438.7 cells/ml among controls (p=0.27). 13 cases and 11

138

controls were on anti-retroviral therapy (ART). All 24 cases were started on ATT and followed

139

up for minimum three months. They all showed significant clinical improvement in the form of

140

relief of symptoms or regression of lymph nodes. None of the patients in control group were

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administered ATT and were kept on follow-up or treated according to alternate diagnosis

142

achieved.

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2. Performance of different techniques for the diagnosis of TBLA (Table 1,2):

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Overall, out of 24 patients of TBLA, the rate of detection was 13/24 (54.16%), 4/24

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(16.67%), 18/24 (75%), 19/24 (79.17%), 16/24 (66.67%) and 22/24 (91.67%) for ZN smear,

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MGIT culture, cytology, LAMP assay, GX and MPCR, respectively. Out of the 6 ‘suspected’

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cases of TBLA in which cytology was inconclusive, 5 were detected as positive for M.

148

tuberculosis by all the molecular techniques i.e. GX, LAMP and MPCR. All these 6 patients

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responded well to ATT on follow-up. All samples from the control group were negative by all

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the methods tested except one for which GX gave a false positive result. Barring GX, the rest of

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the methods had a specificity of 100%. No rifampicin-resistance was detected by GX. On DST,

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all 4 culture-positive cases were susceptible to all four drugs tested.

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Among the different techniques, LAMP assay performed better than GX in not only

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detecting three additional cases missed by GX but also in producing no false-positivity. LAMP

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assay also detected one additional case that was missed on cytology. MPCR further detected

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three more cases missed by LAMP assay. The average turn-around time for different techniques

157

was 1 hour for LAMP, 2 hours for cytology, 3-4 hours for GX and 4-5 hours for MPCR. The

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average cost of running the test, excluding the machine and personnel, was less than $1 for

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LAMP assay and cytology, $10 for GX and $1 for MPCR.

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DISCUSSION

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Despite the availability of numerous diagnostic procedures, there is no gold standard test

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for establishing the diagnosis of TBLA. In the present study 50 samples of lymph node FNAC

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obtained from HIV-infected patients (24 from tubercular lymphadenitis and 26 controls) were

165

subjected to the following tests: ZN smear, MGIT culture, cytology, GX, MPCR and LAMP

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assay. Among the conventional techniques, sensitivity of ZN smear was 54.1%, MGIT culture

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was 16.66% and cytology was 75%. There was no smear or culture or cytology positivity among

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control groups (specificity=100%). Although earlier studies have also reported smear positivity

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ranging from 10 to 50%, culture positivity ranging from 15 to 65 %(18) and cytology ranging

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from 25 to 72% in TBLA cases (19, 20), a direct comparison of the present study with the earlier

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ones may not be correct because the entire patient population in the present study was HIV-

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infected and variable factors like stage of disease, level of immunosuppression and immune-

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reconstitution syndrome may have contributed to the overall yield of detection of M. tuberculosis

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by these conventional methods. The finding that culture positivity in TBLA was much lower

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than that of smear or cytology has been observed earlier also (14, 21, 22). It is attributed to the

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damage caused to M. tuberculosis by the lymphoid-rich tissue in the lymph nodes making the

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bacilli partially devitalized so that they fail to multiply yet retain their staining properties. Higher

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detection yield of 75% was achieved with cytology in the current study. It could be attributed to

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the expertise of the pathologist and the fact that despite being HIV positive, the patients had

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sufficient CD4 counts (mean 347.6 cells/ml) to form epithelioid cell granuloma.

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Among the NAATs evaluated in the present study, LAMP assay using IS6110 gene had a

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sensitivity and specificity of 79.17% and 100%, respectively. Only three studies are available in

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the world literature that have evaluated LAMP for TBLA. Sharma et al(14) have evaluated

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LAMP assay for TBLA using two gene targets, IS6110 and MPB64, and have reported a similar

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sensitivity of 80% for IS6110 alone, while that for MPB64 alone and in combination with IS6110

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was 85.3% and 90%, respectively. While Joon et al (23) have reported a sensitivity and

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specificity of 86.67% and 94.04%, respectively for IS6110-LAMP on a collective of 315 extra-

188

pulmonary samples out of which only ten were from lymph nodes, study by Joon et al (24) has

189

evaluated an alternate gene sdaA. None of the patient population in these three studies was HIV-

190

infected. Previous studies evaluating the utility of LAMP in pulmonary TB as a replacement for

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smear microscopy showed pooled sensitivity of 68% and specificity of 98%. In subgroup of

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patients living with HIV, pooled sensitivity was lower at 64% with specificity of 99%(25).

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The second NAAT evaluated in the current study was GX which gave a sensitivity and

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specificity of 66.67% and 96.15%, respectively. While Pandey et al(26) have reported a similar a

195

sensitivity of GX ranging from 63 to 90% in diagnosing TB from lymph node samples, Ghariani

196

et al (27) and Tadesse et al (28) have reported a higher sensitivity of 87%. There was one false

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positive report of M. tuberculosis by GX in an otherwise confirmed case of lymphoma in the

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present study. This is higher than the previous studies who have reported a specificity ranging

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from 73.3% (22) to 91.1% (28) from FNAC samples of TBLA cases. Although the WHO

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recommendations for the use of GX in HIV-associated pulmonary tuberculosis and in multi-drug

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resistant tuberculosis (MDR-TB) in 2011 were evidence-supported(29), the recommendations

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made in 2014 (30), regarding use of GX for non-respiratory samples especially lymph nodes

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were more conditional based on very low-quality evidence.

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Multiplex PCR, the third NAAT evaluated in the present study, was the test that was

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most sensitive. Using IS6110 and MBP64 as the gene targets, a sensitivity and specificity of

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91.67% and 100%, respectively was achieved in diagnosing TBLA in HIV-infected patients.

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This is in concordance with the earlier study wherein MPCR using the same target genes gave a

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sensitivity of 90% and specificity of 100% from TBLA cases from apparently non-

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immunocompromised patients (7). MPCR using IS6110 and MPB64 genes has reported similar

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sensitivity in detecting other extra-pulmonary pauci-bacillary tuberculosis like tuberculous

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meningitis (83.63%)(15), osteo-articular tuberculosis (82.5%)(31) and gastro-intestinal

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tuberculosis (77.5%)(16).

213

In comparison to other techniques, LAMP assay had a reasonably good sensitivity, better

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than cytology and next only to MPCR. The sensitivity of LAMP assay can be further increased

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by additional gene targets as reported earlier (12), however, the same was not possible in the

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present study. LAMP required much less technical expertise and cost-consuming equipment like

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thermal-cyclers were not needed making it more economical than MPCR. In comparison to GX,

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LAMP was faster, much cheaper and did not give any false-positive result. GX, however, has the

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added advantage of identifying rifampicin resistance and is useful in patients where MDR-TB is

220

suspected. The molecular techniques, however, have the inherent disadvantage of not being able

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to differentiate live bacilli from the dead ones and hence either cytology (showing the tissue

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reaction to tubercle bacilli) or culture (showing actual growth of tubercle bacilli) are needed in

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defining a case of TBLA. In the present study also, cytology performed almost similar to LAMP

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assay and missed only one case that was positive by LAMP assay. This patient, along with all

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other study group patients that were negative by conventional techniques, improved with ATT.

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Hence, in situations where expert pathologist is not available or facilities for sample preparation

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and staining do not exist, LAMP assay can serve as a useful alternative in clinching the diagnosis

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of TBLA in presence of other corroborative evidences.

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Further strength of the study is that all patients were kept on follow up and response of

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patients to antitubercular therapy was monitored. All 24 patients on ATT showed response in

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form of resolution of symptoms/regression of lymph nodes during treatment futher validating the

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test results. No patient in control group developed tuberculosis during study period. Males

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outnumbered females in the present study which could be due to the healthcare-seeking behavior

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which is much higher in men than women in endemic low-resource countries.

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Present study highlights the utility of LAMP assay as a quick and dependable diagnostic

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test for TBLA in HIV-infected patients and may be used in peripheral centers where facilities for

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cytological examination and complex molecular techniques are not available. Further research on

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LAMP assay should focus other pulmonary and extra-pulmonary tuberculosis samples where a

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thorough comparison with established reference standards can be made.

240 241

Conflict of interest: None

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Financial disclosures: None

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Acknowledgement: None

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Table 1. Comparison of different methods for the diagnosis of TBLA in HIV-infected patients. Test

Test

TBLA

Non-TBLA

results

cases

controls

n=24

n=26

Positive

22

0

Negative

2

26

LAMP

Positive

19

0

assay

Negative

5

26

GX

Positive

16

1

Negative

8

25

Positive

18

0

Negative

6

26

Positive

13

0

Negative

11

26

MGIT

Positive

4

0

culture

Negative

20

26

MPCR

Cytology

ZN smear

Sensitivity Specificity

PPV

NPV

%

%

%

%

91.67

100

100

92.85

79.17

100

100

83.87

66.67

96.15

94.11

75.75

75.00

100

100

81.25

54.16

100

100

70.27

16.67

100

100

56.52

Table 2. Performance of different methods for the diagnosis of confirmed and suspected cases of TBLA in HIV-infected patients. ZN MGIT Cytology GX IS6110 MPB6 MPCR LAMP smear cultur PCR 4 PCR e Group I – 11 4 18 13 14 16 17 16 Confirme (61.61% (100% (100%) (72.77%) (77.77%) (88.88) (94.44% (88.88) d TBLA ) ) ) (N=18) Group II – 2 0 0 3 3 4 5 3 Suspected (33.33) (33.33%) (33.33%) (66.66 (83.33% (33.33% TBLA %0 ) ) (N=6) Total 13 4 18 16 17 20 22 19 TBLA (54.16% (16.67 (75%) (66.67%) (70.83%) (83.33 (91.67% (79.17% cases ) %) %) ) ) (N=24)

Figure 1: A panel of microphotographs; a- Smear showing necrotizing granulomatous inflammation (MGG x20X); b- Epithelioid cell granuloma with background showing reactive lymphoid cells (MGG x20X); c- Loose collection of epithelioid cells and reactive lymphocytes (H&E x40X): d- Multinucleated giant cell and lymphocytes (H&E x20X); inset- positive for Acid-fast bacilli (Ziehl-Neelsen stain x100X).

HIGHLIGHTS -

Diagnosing tubercular lymphadenitis (TBLA) in HIV patients is challenging

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Need for simple, accurate, inexpensive test

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GeneXpert has low sensitivity and may give false positive result

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LAMP assay: a suitable alternative for resource-limited countries