1. URINE MYCOBACTERIAL LIPOARABINOMANNAN (LAM) ANTIGEN IN HIV / TB COINFECTED CHILDREN
May Sandar Soe, Chaw Sandar Tun, Khin Nyo Thein
Department of Pediatrics
University of Medicine 2, Yangon

2. Tuberculosis is the most common presenting illness and the major cause of deaths among people living with HIV.
In South-East Asia region there were estimated 4,670,000 new TB cases, 163,000 cases among HIV positive people and 378,000 cases in children.
In Myanmar, estimated 191,000 people were infected with tuberculosis, about 23,000 of them in children, along with 13,000 MDR-TB cases and 18,000 cases co-infected by TB and HIV .
Ref;WHO, Global Tuberculosis Report, 2017

3. Tuberculosis in children is often missed or overlooked.
TB diagnosis is commonly based on clinical symptoms, chest X-ray, microbiological examination and culture.
This is difficult in children because clinical presentation is not specific and chest X-ray interpretation has low accuracy and high inter-observer variation.
Microbiological examination is the gold standard for TB diagnosis, but this is not easy to perform in children due to difficulty in obtaining sputum sample and paucibacillary nature of the disease.
Ref; Reid and Shah, 2009 and Lawn and Wood, 2011

4. It can subsequently be detected in urine and other body fluids.
Urine-based Lipoarabinomannan (LAM) antigen testing has been emerged as a non-invasive alternative approach for diagnosing tuberculosis without reliance on laboratory equipment or reagents.
LAM detection in urine for TB diagnosis was first investigated in the late 1990s.
LAM is a 19 kD (±8.5 kD) lipopolysaccharide, specific to the cell wall of Mycobacterium genus and is released from metabolically active or degrading bacterial cells.
It can subsequently be detected in urine and other body fluids.
Ref: Peter et al, 2012

5. Advantages of urine LAM diagnosis include the ease of specimen collection, short bench-time, low cost and relatively low training and set up requirements.
The test is easy to perform and requires minimal biosafety requirements.
Several studies and a meta-analysis of LAM test have demonstrated improved sensitivity of urinary LAM in the presence of HIV-TB co-infection, which further increases with lower CD4 counts.
WHO recommends that LF-LAM may be used to assist in the diagnosis of tuberculosis in HIV positive patients.

6. RESEARCH OBJECTIVES

7. General objective
To study the urine Mycobacterial Lipoarabinomannan (LAM) antigen in HIV/ TB coinfected children 
Specific objectives
To find out the positivity of urine Lipoarabinomannan (LAM) test in HIV/ TB coinfected children
To determine the positivity of AFB smear microscopy in HIV/ TB coinfected children
To assess the positivity of Xpert MTB/RIF in HIV/ TB coinfected children

8. MATERIALS AND METHODS

9. The study was conducted in inpatient and outpatient department of Mingalardon Specialist Hospital, Yankin Children Hospital and Tharkayta Specialist Hospital.
This study was hospital based cross sectional descriptive study and the study period was one year from 1st January 2018 to 31th December 2018.

10. 40 cases of 5 to 15 years’ age clinically diagnosed pulmonary or extrapulmonary TB in HIV infected children were included.
Children who had previously diagnosed to have pulmonary or extra pulmonary TB and already received anti-TB medications for more than two weeks and who had either leukocyturia, hematuria or moderate to severe proteinuria (>3+) reflecting renal abnormalities in urinalysis were excluded.

11. RESEARCH PROCEDURE

14. Statistical Analysis
Data collection was done by proforma.
Data entry, data clean up, data summarization and data analysis were carried out by computer using statistical package for social science (SPSS) software version 16.
Descriptive and summary statistics were carried out.

15. For the continuous data, mean, median and standard deviation were calculated.
For the categorical data, difference between proportions of sample variables was expressed as number and percentage.
Graphs and charts were displayed for clear presentation.
Data was statistically analyzed with test of significance and P value of <0.05 was considered significant.

16. Ethical considerations
This study was approved by the Ethics Review Committee of University of Medicine 2, Yangon.
Informed consent was taken from one of the parents or guardians to commencement of this study.

17. RESUITS

18. Table 1. Age distribution of study population
Age(in year)
Number
Percentage
5-<8 years 22
55.0
8-15 years 18
45.0
Total 40
100.0

19. Table 2. Sex distribution of study population

20. Table 3. WHO clinical staging of HIV/TB coinfected children

21. Table 4. WHO immunological staging of HIV/TB coinfected children
25%
27.5%
7.5%

22. Table 5. Distribution of different type of TB in HIV infected children
Types of TB Disease
Frequency
Percent
Primary complex 16
40.0
Pulmonary TB
TB pleural effusion 3
7.5
TB Meningitis 1
2.5
TB Lymphadenitis
TB abdomen 2
5.0
TB pericardial effusion
Total 40
100.0

23. Table 6. Result of urine LAM assay in HIV/TB coinfected children
Number
Percent
Positive 10
25.0
Negative 30
75.0
Total 40
100.0

24. Table 7. Result of AFB smear microscopy in HIV/TB coinfected children
Number
Percent
AFB positive 2
5.0
AFB negative 38
95.0
Total 40
100.0

25. Table 8. Result of Xpert MTB/RIF assay in HIV/TB coinfected children
Number
Percent
Positive 2
5.0
Negative 38
95.0
Total 40
100.0

26. Table 9. Association between AFB smear microscopy and urine LAM assay in HIV/TB coinfected children
Urine LAM positive
Urine LAM negative
Total
AFB positive 2
AFB negative 8 30 38 10 40

27. Table10. Association between Xpert MTB/RIF assay and urine LAM assay in HIV/TB coinfected children
Urine LAM positive
Urine LAM negative
Total
X pert positive 2
X pert negative 8 30 38 10 40

28. Table11. Association between clinical staging and urine LAM assay in HIV/TB coinfected children

29. Table 12. Association between immunological staging and urine LAM assay in HIV/TB coinfected children

30. DISCUSSION

31. The male and female ratio was 1:1.22.
No significant influence of age or sex on test positivity could be demonstrated.
Pulmonary tuberculosis cases were more common than extrapulmonary tuberculosis in this study.
In the present study, tuberculosis infection in HIV infected children were most common in clinical stage III group and severe immunological stage group (CD4 counts less than 200 cells/mm3).

32. Kroidl et al, 2015 conducted a prospective observational study
performance of urine LAM assays in 6 weeks to 14 years age of pediatric tuberculosis, at OPD of the Mbeya Zonal Referral Hospital, Tanzania
showed HIV positive children had urine LAM positive in five out of ten tuberculosis confirmed cases (50%) ,one out of eighteen probable tuberculosis cases (6%) and no urine IAM positive in tuberculosis excluded cases

33. A study of urine LAM testing for diagnosis of pulmonary tuberculosis in <15 years children done by Mark et al, 2014 at a primary health-care clinic and pediatric referral hospital in Cape Town, South Africa, showed 25 out of 106 HIV positive patients (24%) were urine LAM test positive.
Sylvia et al, 2018 done a study in Kenya among 137 hospitalized children in aged <12 years who were HIV-infected cases showed fifteen (11 %) were urine LAM positive.

34. Although, there was no previous data for pediatric urine LAM test in Myanmar, there was an adult study.
A study, clinical utility of the urine-based lateral flow LAM assay in HIV- infected adults in Myanmar was done by Swe-Swe-Thit (2017) at Insein General Hospital from 1st July 2015 to 31st December 2015.
That study revealed the baseline LF-LAM test was positive in 201 patients of 517 HIV infected cases (39%).

35. In the present study, urine LAM was positive in ten patients among forty HIV /TB coinfected children (25%).
This study provided evidence to support WHO’s recommendation that urine LAM is helpful for tuberculosis diagnosis in HIV-infected children.

36. Higher yield of urine LAM testing was reported among individuals with low CD4 cell counts and more advanced HIV disease (Shah et al. 2009; Peter et al.,2012; Talbot et al. 2012).
LAM test positivity correlated with the degree of immunosuppression and also more sensitive inpatients with lower CD4 cell counts (typically <100cells/μl) (Minion J, 2011).

37. This was in concordance with the present study which showed that urine LAM positive were in clinical stage III and IV group.
In WHO immunological staging group, the most urine LAM positivity were in severe immunological stage.
The finding similar to this study was occurred in the Myanmar adult study done by Swe-Swe-Thit (2017) showed that the patients with positive LF- LAM test were more likely to have advanced immunodeficiency (CD4 T- cell count < 100 cells/mm3).

38. The study conducted in Tanzania (Kroidl et al, 2015) stated that urine LAM test positive in 6 weeks to 14 years of pediatric age group were 38% in none or mild immunological stages and 15% in advanced or severe immunological stages.
The comparison of the performance of LAM-assays in children with advanced or severe immunosuppression versus those with mild or no immunosuppression does not suggest a higher sensitivity of LAM diagnostics in those with advanced HIV infection.

39. LAM detection results increase with bacterial burden (Shah M et al,2010).
The LAM positive have been shown to be more often in smear positive than in smear negative patients (Mutetwa et al,2009).
In the present study, smear positive and gene Xpert positive results were only 5% but occurred in different patients.

40. Although urine LAM positive were 100% in those smear positive and gene Xpert positive patients, Fisher's Exact Test, p was
Therefore, there is no statistical association between AFB smear microscopy or Gene Xpert assay and urine LAM in HIV/TB coinfected children.
Whatever the explanation for the disappointing results, microbiological examination usually shows as low positivity in children due to paucibacilarity of the disease (Marais et al,2007).

41. In adult study done by Taye et al,2014 in the Oromia region of Ethiopia stated that AFB were detected in sputum from 30 of 757 participants and LAM was detected in urine from 78 patients.
The author suggested that a combination of sputum smear microscopy and determine TB-LAM test might increase diagnostic accuracy.

42. The recent tuberculosis diagnostic accuracy study conducted in HIV-infected children in Kenya, urine LAM performance (compared with sputum/gastric aspirate Xpert or culture) improved among children with severe immunosuppression (LaCourse SM et al, 2018).
The study in Tazania showed the sensitivity of combining LAM diagnostic tests with either smear microscopy or Xpert MTB/RIF-assay was better than each diagnostic test alone (I.Kroidl et al, 2015).

43. CONCLUSION

44. This study has pointed out the possible use of the urine LAM test, in the diagnosis of TB in patients with advanced HIV infection.
Urine LAM test is a rapid test, easy to perform and requires minimal biosafety requirements so rapid diagnosis of active TB in HIV infected children may be achieved.
The combination of LAM tests with other TB diagnostic tools could substantially improve the detection of TB in HIV co-infected children.
Therefore, it can be concluded that urine LAM tests is available test to assist the diagnosis of tuberculosis in HIV infected children especially in advanced HIV disease clinically as well as immunologically.

45. ACKNOWLEDGEMENTS

46. I would like to express my heartful thanks to Professor Aye Tun, Professor Khin Nyo Thein , Senior Consultant Dr. Chaw Sandar Tun, Associate Professor, Dr. Kyi Thar Myo Wynn, Senior Consultant, Dr. Aye Aye Khaing, Senior Consultant, Dr. Myo Khaing and also to all who were helped in conduction of this study including participated patients and their parents.

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51. THANKS