Determine TB –LAM Lateral Flow Assay, Does M. tuberculosis lineage influence its performance?

doi:10.21203/rs.2.9331/v1

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Determine TB –LAM Lateral Flow Assay, Does M. tuberculosis lineage influence its performance?

https://doi.org/10.21203/rs.2.9331/v1

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Background

The World Health Organization conditionally recommended the Determine lateral flow TB-LAM (LF-LAM) assay for tuberculosis diagnosis. The test is recommended for diagnosis and screening of active tuberculosis among HIV-infected individual’s adults with signs and symptoms of TB (pulmonary and/or extra pulmonary) with CD4 cell count less than or equal to 100 cells/mm3 or HIV-positive patients who are seriously ill, regardless of CD4 cell counts. However, the effect of MTB genetic diversity to the performance of LF-LAM remains unknown. We determined the perfomance of the against M. tuberculosis (MTB) lineages among HIV-positive individuals with pulmonary and/or MTB-bacteremia.

Methods

In a retrospective study, of 68 patients culture-positive for MTB, we used spoligotyping and/or MIRU-VNTR methods to determine the MTB-lineages. Patients 51 had both pulmonary and MTB-bacteremia and 17 patients had exclusively PTB. We compared the performance of LF-LAM against MTB-lineage in blood and sputum samples. LF-LAM performance was also analyzed in presence of mixed MTB-infection and patient CD4 cell count.

Results

The median age (interquartile range; IQR) of participants was 32 (30-35) and only sixty-six patients had CD4 results. The median (IQR) CD4 cell count/mm3 was 27(18-34). MTB-lineages in sputum were; CAS (L3) 17/68 (25%) and Euro-American-lineage (L4) 55/68 (75%) whereas in blood were L3 7/51 (13.7%) and L4 44/51 (86.3%). Among patients with TB-bacteraemia, LF-LAM was positive 100% for L3 and 32/44 (73%) for L4. LF-LAM was 100% positive among exclusively PTB patients with L3 and L4. The results did’not change by presence of mixed MTB-infection. Categroizing patients as CD4 <50 and ≥50 cell/mm3. All 100% of L3 strains remained with a positive LF-LAM test in both categories whereas 11 (68.7%) and 30 (82%) of the patients with CD >50 and ≤50 cells/mm3 respectively, with L4 had a positive LF-LAM test.

Conclusions

Our study show that L3 may have more specificity for LF-LAM assay than L4. Regional diversity in performance of LF-LAM assay may occur depending on the dorminat MTB lineage. Future studies on a large sample size with more MTB-lineage diversity are recommended.

Internal Medicine Specialties

LF-LAM

MTB Lineages

HIV

Atypical presentation of HIV-related tuberculosis is common among individuals with CD4 less than 200 cells/mm3. TB is usually disseminated and smear negative. In 2013, WHO recommended the use of Xpert MTB/RIF test as a frontline test for HIV-related and some forms of extra-pulmonary TB (EPTB) [1], although data about the Xpert performance in EPTB are limited. A point of care non-sputum based test that is capable of diagnosing all forms of TB is desirable [2].

Hypothetically, in disseminated TB disease, part of the M. tuberculosis cell wall is excreted in urine as lipoarabinomannan (LAM), which can be detected, although detecting LAM of the whole organism from renal TB disease, given the Xpert positivity has been hypothesised[3]. The “Determine TB LAM Ag test” is a point-of-care lateral flow immune-chromatographic urine strip test (LF-LAM; Alere Inc, Waltham, Massachusetts) detects LAM in the urine of a patient and offers results in 20 minutes. This test was endorsed by WHO in 2015, for diagnosis and screening of active tuberculosis among HIV-infected individuals adults with signs and symptoms of TB (pulmonary and/or extra pulmonary) with CD4 cell count less than or equal to 100 cells/mm3 or HIV-positive patients who are seriously ill, regardless of CD4 cell counts[4]. However, data about the performance of LAM test in different strains of M. tuberculosis is still lacking.

Studies have associated some M. tuberculosis lineages with EPTB more than others [5]. Data from animal models suggest that the structure, internal heterogeneity and abundance of LAM vary between M. tuberculosis strains exhibiting distinct degrees of virulence [6-8]. In Uganda, the most prevalent M. tuberculosis lineages are the Euro-American lineage, lineage 4 (L4) and the Central Asian Strain (CAS), lineage 3 (L3) [9]. L4 strains have been found to have faster intracellular growth whereas M. tuberculosis lineage 3 (L3) have slower growth compared to wild-type M. tuberculosis H37Rv[10]. M. tuberculosis lineages differ significantly in their mycolic acid (MA) profiles with MA profiles of L4 similar among sub family members but different when compared to other main lineages such as L1, L2 and L3 [11]. Data about the performance of LAM against different M. tuberculosis strains may explain differences in the performance of the Determine TB LAM tests in different regions where different M. tuberculosis strains may dominate.

We retrospectively reviewed LF-LAM results from our previous study [12] and M. tuberculosis lineage data from our previous studies [12, 13]. The aim was to understand whether M. tuberculosis lineage and mixed infection among concurrent M. tuberculosis isolated from sputum and/or blood compartment influence performance of LF-LAM assay among HIV-positive individuals with advanced immunosuppression.

Patient population and information were collected as described in the main study [12]. Briefly, from a prospective cohort of HIV-positive adult aged 18 years and above from outpatients’ department of the Infectious Diseases Institute [IDI] and inpatients of Mulago National Referral Hospital medial wards, both in Kampala, Uganda. Participants were tested for both pulmonary and extra pulmonary tuberculosis using laboratory culture methods as described in the previous study [13]. A total of 68 HIV-positive patients with either pulmonary tuberculosis and/or TB bacteremia were considered in the current study. Briefly, participant’s sputum samples were cultured using mycobacterial growth indicator tube (MGIT 960) in a MGIT machine and incubated for up to six weeks. The same sample was also inoculated on Lowenstein Jensen (LJ) media and incubated at 37oc for up to eight weeks following standard protocol whereas blood samples were cultured in an automated machine (BACTEC 9120), incubated up to six weeks and reported as previously described [12]. The M. tuberculosis isolates were tested for susceptibility to anti-TB drugs also as previously described [13]. Only three participants with isolate pairs from sputum and blood showed resistance to ethambutaol in MGIT 960 at 5.0 µg/mL, all others were susceptible to first-line TB drugs.

Isolates were typed for M. tuberculosis lineages using Mycobacterial Interspersed Repetitive Units Variable Number of Tandem Repeats (MIRU-VNTR) 24 loci, and/or spoligotyping (spacer oligonucleotide typing) performed on bacterial lysates as described in our previous study [13]. For lineage assignment, we considered either method and where there was disagreement, MIRU-VNTR-24 loci was used as final results if available. MIRU-VNTR-24 loci results with more than one allele at a given locus were considered to have mixed infection, were considered clonal variants.

Urine samples were also processed and tested using Determine TB-LAM lateral flow strips also as described elsewhere [12]. The LF-LAM assay was performed by two independent laboratory technicians blinded to all clinical information and other diagnostic results. A reading grade intensity greater than or equal to 1 was considered as a positive result.

For statistical analysis, Stata version 13 (StataCorp LP, 4905, Lakeway drive College Station, TX, 77845, USA) was used and confidence intervals for proportions were calculated.

Ethical approvals for the main study were obtained from the Joint Clinical Research Center Institutional Review Board (IRB), Kampala, Uganda and from the Uganda National Council of Science and Technology (UNCST).

A total of 68 of the participants of which 51 had both pulmonary tuberculosis and M. tuberculosis bacteraemia and 17 with exclusively pulmonary tuberculosis were evaluated. The median age (Inter-quartile range; IQR) of participants was 32 (30-35) and only sixty-six patients had CD4 results with a median (IQR) CD4 cell count/mm3 of 27 (18-34)

From sputum samples; 13/68(19.1%) were L3 and 55/68 (80.9%) were L4. Among the patients with TB bacteremia, M. tuberculosis lineages in sputum sample, LF-LAM was positive in all the seven samples with L3 7 (100%) and among the 44 samples with L4, 32(72.7%) positive, 4(9.1%) negative and 8 (18.2%) indeterminate. Among patients with exclusively pulmonary tuberculosis, six (100%) of the L3 and 11(100%) of the L4 were LF-LAM positive.

LF-LAM grades for positives were; 1+ 10 (14.7%), 2+ 12 (17.6%), 3+ 25(36.7%) and 4+ or greater 9 (13.2%). M. tuberculosis lineages from blood samples were; the CAS (L3) 10/51 (19.6%) and Euro-American lineage (L4) 41/51 (80.4%).

Among the 10 participants with L3 in blood samples, LF-LAM was; negative in none, but indeterminate in one patient. Whereas among the 41 participants with lineage 4 in blood, LF-LAM was; negative among 4 (9.8%; 95% CI, 2.7% -23.1%), positive among 30 (73.2% 95% CI, 57.0%-85.7%) and indeterminate among 7 (17.1%, 95% CI, 7.1%-32.0%), Table 1.

Analysis of the M. tuberculosis typing data, 2/68 (2.9%) had mixed lineages one with L4 in blood and L3 in sputum and the other with opposite results. Stratification by CD4 cell count (CD4 >50 cell/mm3 and CD4 ≤ 50 cell/mm3) did not change the findings, Table 2. All 100% of L3 strains remained with a positive LF-LAM test in both categories whereas 11 (68.7%) and 30 (82%) of the patients with CD >50 and ≤50 cells/mm3 respectively, with L4 had a positive LF-LAM test.

Our study shows high level of LF-LAM positivity for Central Asian Strain (CAS) lineage (L3) compared to the Euro-American lineage (L4). The findings further demonstrate unexpectedly high indeterminate LF-LAM results among patients with L4 (contributed almost all the indeterminate results) compared to L3 and all negatives were reported among those with L4. These findings remained similar irrespective of which sample (blood or sputum; bacteremic or non-bacteremic) from which the M. tuberculosis lineage were typed and CD4 cell count levels.

Mixed infection with M. tuberculosis strains of the same sub lineage showed no effect, whereas mixed infection with strains from different sub-lineage in different compartment indicated an effect on LF-LAM results. The high levels of LF-LAM positivity could be explained by the fact that studies have documented a difference in M. tuberculosis phylogenetic lineage distribution by site of infection and L3 found to be more frequent in extra pulmonary tuberculosis compare to L4 [5]. This suggests the polyclonal antibodies may recognize different sub-strains of LAM differently.

Furthermore, a review study showed LAM, and its related glycolipids to induce divergent and opposing immune responses to M. tuberculosis depending on structural diversity and experimental variations [8]. A study profiling mycolic acid biosynthesis showed that the strain genetic background influences mycolic acid metabolism indicating that M. tuberculosis lineages may be vital during designing and validation of novel diagnostic tool [11]. Indeed, the same study found that mycolic acid profiles for L4 was different from that of L3. The predominance of L3 in EPTB together with the difference in mycolic acid composition may explain the high levels of indeterminate results seen for LF-LAM assay among participants with L4. The effects of mixed strain infection to the performance of LF-LAM require more deep investigation. Our study findings may inform future studies towards policy and practices for deployment of this novel diagnostic. For example, the cut-off criteria for use of LAM may apply differently in regions depending on the predominant M. tuberculosis lineages. Such implication may include; LF-LAM having more sensitivity in areas where L3 is predominant and hence the cut-off for CD4 cell count/mm3 among HIV-positive individuals as per WHO guideline may be greater than 100 cell/mm3 in such areas and less than 100 cell/mm3 in areas where L4 predominates. An example of such implication includes the rapid identification assay for M. tuberculosis complex that identifies MPT64 antigen of the M. tuberculosis complex that has been deemed unfit in areas predominated by M. africanum (MAF; L7) [14, 15]. Our findings of all L4 being LF-LAM positive among the exclusively pulmonary TB patients raise a hypothesis that may be during dissemination, L4 loses a structural component which is vital for LF-LAM positivity and suggests for more studies.

Our study had some limitations; consistent with previous studies [16], L4 is the most dominant M. tuberculosis lineage in this region, Kampala and hence our study had little M. tuberculosis diversity to compare in our study population, however, this being the first of the studies evaluating this phenomenon, our findings will significantly inform future studies. M. tuberculosis typing on culture may have excluded minority groups of M. tuberculosis lineages in presence of mixed strain infection thereby leading to recovery of only dominant population strain[17]. However, since typing was done on primary culture isolates this is expected to be of a minimal effect. The unique study population with both pulmonary and M. tuberculosis bacteremia and due to fact that LF-LAM was found to be more sensitive in patients with M. tuberculosis bacteremia constitutes the strength of our study findings.

our study has showed that M. tuberculosis lineage 3 offers higher LF-LAM positivity compared to M. tuberculosis lineage 4. Strain factors leading to higher numbers of LF-LAM assay indeterminate results require more investigations in a larger study with more M. tuberculosis lineage diversity.

MTB M. tuberculosis

LF-LAM Lateral Flow lipoarabinomannan

PTB Pulmonary tuberculosis

MIRU-VNTR Mycobacterial Interspersed Repetitive Units Variable Number of Tandem Repeats

IDI Infectious Diseases Institute

Ethics approval and consent to participate

Consent for publication

Not applicable

Availability of data and material

All data generated or analysed during this study are included in this published article and its supplementary information file

Competing interests

The authors declare that they have no competing interests

Funding

The typing work was supported by the Specific Grant Agreement 2013-1479, Erasmus Mundus Joint Doctorate Fellowship to WS. WS is a NURTURE fellow under NIH grant D43TW010132. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author’s contributions: WS contributed to the conception and design of the study, WS, MLJ, LN, YCM EK, acquisition of data, analysis and interpretation of data, WS drafting the article, YCM, LN revising it critically for important intellectual content, all authors gave final approval of the version to be submitted.

Acknowledgements. The authors acknowledge the clinic and laboratory staff at the Mycobacteriology laboratory as well as at the Infectious Diseases Institute at the College of Health Sciences, Makerere University.

1. WHO: World Health Organization; Tuberculosis diagnostics, Xpert MTB/RIF Test. WHO Recommendations October 2013; Available: Accessed Aug 1 2014. 2014.

2. WHO: World Health Organization. Global tuberculosis report 2014. http://apps.who.int/iris/bitstream/10665/137094/1/9789241564809_eng.pdf Accessed September 26-2015. 2014.

3. Lawn SD, Gupta-Wright A: Detection of lipoarabinomannan (LAM) in urine is indicative of disseminated TB with renal involvement in patients living with HIV and advanced immunodeficiency: evidence and implications. Trans R Soc Trop Med Hyg 2016, 110(3):180-185.

4. WHO: World Health Organization 2015: The use of lateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosis and screening of active tuberculosis in people living with HIV; Policy guidance. Available at; http://www.who.int/tb/areas-of-work/laboratory/policy_statement_lam_web.pdf. Acessed January 16th 2016. 2015.

5. Click ES, Moonan PK, Winston CA, Cowan LS, Oeltmann JE: Relationship between Mycobacterium tuberculosis phylogenetic lineage and clinical site of tuberculosis. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2012, 54(2):211-219.

6. Gaur RL, Ren K, Blumenthal A, Bhamidi S, Gonzalez-Nilo FD, Jackson M, Zare RN, Ehrt S, Ernst JD, Banaei N: LprG-mediated surface expression of lipoarabinomannan is essential for virulence of Mycobacterium tuberculosis. PLoS pathogens 2014, 10(9):e1004376.

7. Pan Q, Wang Q, Sun X, Xia X, Wu S, Luo F, Zhang XL: Aptamer against mannose-capped lipoarabinomannan inhibits virulent Mycobacterium tuberculosis infection in mice and rhesus monkeys. Mol Ther 2014, 22(5):940-951.

8. Kallenius G, Correia-Neves M, Buteme H, Hamasur B, Svenson SB: Lipoarabinomannan, and its related glycolipids, induce divergent and opposing immune responses to Mycobacterium tuberculosis depending on structural diversity and experimental variations. Tuberculosis 2016, 96:120-130.

9. Wampande EM, Mupere E, Debanne SM, Asiimwe BB, Nsereko M, Mayanja H, Eisenach K, Kaplan G, Boom HW, Gagneux S et al: Long-term dominance of Mycobacterium tuberculosis Uganda family in peri-urban Kampala-Uganda is not associated with cavitary disease. BMC infectious diseases 2013, 13:484.

10. Homolka S, Niemann S, Russell DG, Rohde KH: Functional genetic diversity among Mycobacterium tuberculosis complex clinical isolates: delineation of conserved core and lineage-specific transcriptomes during intracellular survival. PLoS pathogens 2010, 6(7):e1000988.

11. Portevin D, Sukumar S, Coscolla M, Shui G, Li B, Guan XL, Bendt AK, Young D, Gagneux S, Wenk MR: Lipidomics and genomics of Mycobacterium tuberculosis reveal lineage-specific trends in mycolic acid biosynthesis. Microbiologyopen 2014, 3(6):823-835.

12. Nakiyingi L, Moodley VM, Manabe YC, Nicol MP, Holshouser M, Armstrong DT, Zemanay W, Sikhondze W, Mbabazi O, Nonyane BA et al: Diagnostic accuracy of a rapid urine lipoarabinomannan test for tuberculosis in HIV-infected adults. Journal of acquired immune deficiency syndromes 2014.

13. Ssengooba W, Cobelens FG, Nakiyingi L, Mboowa G, Armstrong DT, Manabe YC, Joloba ML, de Jong BC: High Genotypic Discordance of Concurrent Mycobacterium tuberculosis Isolates from Sputum and Blood of HIV-Infected Individuals. PloS one 2015, 10(7):e0132581.

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Table 1: Performance of LF-LAM assay by M. tuberculosis lineage isolated from blood and/or sputum among HIV-positive individuals

Parameter	M. tuberculosis lineage in blood		M. tuberculosis lineage in sputum		M. tuberculosis lineage in bacteremic patients		M. tuberculosis lineage in non-bacteremic patients
Lineage	L3 n (%) n=10	L4 n (%) n=41	L3 n (%) n=13	L4 n (%) n=55	L3 n (%) n=7	L4 n (%) n=44	L3 n (%) n=6	L4 n (%) n=11
LF-LAM
Positive	9 (90)	30 (73.2)	13 (100)	43 (78.2)	0	32 (72.7)	6 (100)	11 (100)
Negative	0	4 (9.8)	0	4 (7.3)	7 (100)	4 (9.1)	0	0
Indeterminate	1 (10)	7 (17.1)	0	8 (14.5)	0	8 (18.2)	0	0
LF-LAM grade
1+	1 (10)	9 (22)	1 (7.7)	9 (16.4)	1 (14.3)	9 (20.4)	0	0
2+	1 (10)	6 (14.6)	3 (23.1)	9 (16.4)	1 (14.3)	6 (13.6)	2 (33.3)	3 (27.3)
3+	4 (40)	13 (31.7)	6 ( 46.1)	19 (34.5)	3 (42.8)	14 (31.8)	3 (50.0)	5 (45.4)
≥4+	3 (30)	2 (4.9)	3 (23.1)	6 (10.9)	2 (28.6)	3 (6.8)	1 (16.7)	3 (27.3)

L3 = M. tuberculosis Lineage 3, L4= M. tuberculosis Lineage 4

Table 2: Stratification of LF-LAM performance by CD4 cell count and M. tuberculosis lineages

	CD4 >50 cell/mm3		CD4 ≤ 50 cell/mm3
LF-LAM	L3 n (%)	L4 n (%)	L3 n (%)	L4 n (%)
Negative	0	2 (12.5)	0	2 (5.4)
Positive	5 (100)	11(68.7)	8 (100)	30 (82)
Indeterminate	0	3 (18.8)	0	5 (13.5)
Total	5	16	8	37

L3 = M. tuberculosis Lineage 3, L4= M. tuberculosis Lineage 4

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Determine TB –LAM Lateral Flow Assay, Does M. tuberculosis lineage influence its performance?

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Abstract

Background

METHODS

RESULTS

DISCUSSION

Conclusions

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REFERENCES

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