Cerebrospinal Fluid Lipoarabinomannan Lateral Flow Assay in Diagnosing Tuberculous Meningitis in HIV-Infected Patients

Sabai Phyu, Kyaw Swar Lin, Kyi Phyu Aye, Aye Su Mon, Nay Win Aung, Wah Wah Aung & Htin Aung Saw

Special Issue May 2020

Positivity of diagnostic tools among study population

Of 279 clinically-diagnosed TBM patients, 26(9.3%) were CSF Xpert MTB/RIF positive, 24(8.6%)were CSF Mycobacterium tuberculosis culture positive and 26(9.3%) were CSF LAM-LFA positive. Fifteen were both Xpert MTB/RIF and culture positive, 9 were culture positive/Xpert MTB/RIF negative and 11 were culture negative/Xpert MTB/RIF positive.Thirty-five cases (12.5%) were bacteriologically confirmed as definite TB Meither by gold standard tests, CSF Xpert MTB/RIF and/or culture positivity.

Demographic and clinical characteristics of definite TBM patients (n=35)

Demographic data, related clinical data and routine laboratory result of CSF in definite TBM cases of the HIV-associated TBM patients (n=35) are shown in Table 1.

Comparison of LAM test with gold standard tests

Among 35 definite TBM cases, 15(57.69%) cases were LAM positive and 20(7.91%) cases were LAM negative. Eleven cases of negative result in gold standard tests showed LAM Ag positivity (Table 2).

 Table 1. Demographic data, related clinical data and routine laboratory result of CSF in definite HIV- associated TBM patients (n=35)

LAM test	Gold standard result		Grand total
	Positiven(%)	Negativen(%)
Positive	15(42.68)	11(4.51)	26(9.32)
Negative	20 (57.14)	223(95.49)	253(90.68)
Grand total	35(100)	244(100)	279(100.00)

Mortality outcome in relation to CD4+ count and to CSF pressure

Mean CSF pressure of 35 patients with definite TBM was 60.55+/-103 mmH2O. HIV patients who had CSF pressure <120 mmHg and CD4+ count of less than 100 cells/μl were significantly more likely to suffer mortality (p=0.04 and p=0.36), respectively (Table 3).

 Table 3. Mortality outcome in relation to CD4+countand CSF pressure

Crude OR=6.15 (95% CI=0.01-1.8), p=0.04(mortality in relation to CSF pressure), Crude OR=0.36 (95% CI=0.08-1.69), p=1.68(mortality in relation to CD4+count)

 

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Tuberculous meningitis (TBM) occurs more commonly in HIV-infected individuals than in HIV-uninfected individuals and still an ongoing problem in HIV-infected patients in developing countries.¹ In 2017, an estimated 10 million people developed tuberculosis (TB) and 1.2 million were coinfected with HIV. TB-HIV-coinfected patients have an increased risk of developing extrapulmonary TB (EPTB).² TBM is the most severe form of EPTB, with in-hospital mortality rate of 13 to 72%. Prompt treatment with anti-TB drugs improves patient outcome. Therefore, sensitive and rapid diagnostic tests to diagnose TBM are vital. Diagnosis of TBM is made through microscopic visualization of acid-fast bacilli (AFB) or culturing Mycobacterium tuberculosis in cerebrospinal fluid (CSF). The sensitivity of AFB microscopy in CSF is highly variable (0% to 87%) irrespective of HIV and depends on case definition, the amount of CSF, and the experience of the technician. In resource-limited settings, the clinical use of culture is limited by availability, long turnaround time, laboratory safety issues, and high cost.³

The Xpert MTB/RIF assay (Cepheid, CA, USA) is a commercial nucleic acid amplification test that uses real-time PCR to detect M. tuberculosis and identify rifampin resistance. The World Health Organization (WHO) recommends the use of Xpert MTB/ RIF rather than conventional microscopy and culture as the initial diagnostic test for CSF specimens in patients suspected to be harboring TBM.⁴ Xpert MTB/RIF in CSF has sensitivity of 80% and specificity of 99%, based on pooled data from 13 studies, including 709 CSF samples; a minority of these samples were from HIV-infected patients.⁵

Therefore, TBM is often diagnosed pre-sumptively on the basis of clinical, basic laboratory and radiological findings in clinical practice. Now WHO recommended urinary LAM antigen test to assist in diagnoy of TB. Lipoarabinomannan (LAM), a M. tuberculosis cell wall component, can be detected in multiple bodily fluids, including CSF of TB patients.⁶ 

A LAM enzyme-linked immunosorbent assay (ELISA) (Clearview TB ELISA; Alere, Waltham, MA, USA) developed for urinary LAM detection was used in studies of CSF, an off-label use, in South African cohorts of clinical meningitis patients. The studies reported sensitivity of 64% to 69% and specificity of 62% to 65% for diagnosis of culture - or PCR-positive TBM. Higher sensitivity and specificity were noted in HIV-infected patients with CD4 cell counts below 100 cells/mm.^{4, 7}

Recently, a point-of-care lateral flow assay (LFA) was developed (Determine TB LAM; Alere, Waltham, MA, USA) that detects LAM in unprocessed urine.⁶ This test offers important advantages over the ELISA in terms of speed, simplicity, and costs. LFA-LAM could be the urgently needed rapid test for TBM. However, its use in CSF for the diagnosis of TBM is not yet investigated and data on CSF LAM-LFT and Xpert MTB/RIF performance in high-prevalence-TB and -HIV settings are limited. The present study was carried out to detect proportion of TBM among HIV patients attending three specialist hospitals in Yangon and to determine the clinical characteristic of HIV- associated tuberculous meningitis patients and to evaluate usefulness of point-of-care CSF LAM-LFA to diagnose those cases.

MATERIALS AND METHODS

Study design and study period

A cross-sectional descriptive study was carried out during 2017-2018.

Study population 

A total of 279 HIV patients with clinically diagnosed TBM attending Waibagi, Thakayta and Mingaladon Specialist Hospitals during the study period.

Inclusion criteria 

HIV-seropositive patients with clinically diagnosed TBM (both male and female, 18 years age) who gave written informed consent.

Exclusion criteria 

Critically ill patients, patients with signs of intracranial space occupying lesions or local infection in lumbar space 

Socio-demographic and clinical data 

After taking the informed consent, the relevant socio-demographic data and clinical data regarding the symptoms and duration of disease, previous history of anti-TB treatment and duration, concomitant diseases, history of HIV infection, investigations such as CD4+ count, Xpert MTB/RIF results, other laboratory tests etc., were collected. 

Laboratory procedures 

Sample collection

After obtaining informed consent, demographic and clinical data had been noted in proforma. Lumber puncture was carried out under aseptic condition according to the standard procedure and CSF pressure was measured by Spinomanometer. CSF was collected by three sterile-leak proof tubes, one for routine CSF analysis and microscopy, second tube for routine Xpert MTB/RIF assay and the extra tube about 0.5 ml for LAM-LFA and TB culture.

CSF LAM-LFA

One drop (about 60 ul) of CSF was applied to LAM-LFA test strip (Lateral-flow TB LAM antigen testing, Alere, Waltham, MA, USA). After 15 minutes, 2 experienced laboratory technicians independently read the test strip by comparing the LFA test result to the manufacturer-supplied reference card. The results were calculated the sensitivity, specificity, PPV, and NPV of LAM lateral flow antigen test for definite TBM, using no TBM as the denominator.⁸

CSF routine examination, microscopy and CSF Xpert MTB/RIF  

Routine examination of CSF (eg. protein, sugar, cells count, Ziehl-Neelsen staining and Indian ink staining) was carried out. CSF was also tested by Xpert MTB/RIF to detect TB and rifampicin resistance. The laboratory results were recorded in the proforma.

CSF Mycobacterium tuberculosis culture and anti-TB drug susceptibility testing (DST)

The CSF samples were inoculated onto Lowenstein-Jensen solid medium and incubated at 37°C. Tubes were checked at weekly intervals until 12 weeks for the colonial morphology and growth reading.9 DST of first-line anti-TB was performed by solid culture-based proportion method and DST of pyrazinamide and second line anti-TB drugs was performed by liquid culture-based MGIT system.10 Definite TBM was defined as culture positive for TB and/or a positive CSF Xpert MTB/RIF result.

Statistical analysis

The data were analyzed using SPSS version 23 statistical software. Statistical comparison between various groups was made using Fisher’s exact test when appropriate and their 95% confidence interval were used to measure the strength of associations. The level of significance was set as p0.05.

Ethical consideration 

This study was approved by the Protocol Academic Board, Department of Medical Research.

Firstly a newly developed clinical prediction rule, suited to resource-poor high HIV prevalence settings, is a useful rule-in test for the rapid diagnosis of TBM. LAM antigen, which has not previously been prospectively evaluated in CSF, is useful as a rapid rule-in test for the diagnosis of TBM in HIV-infected individuals with advanced immunosuppression. Thus, combining the prediction rule with LAM antigen detection further increases the rule-in value for TBM.⁵

The initial evaluation of patient suspect of TBM should always include chest X ray, ultrasound, CD4 count and CSF examination. Diagnostic yield of acid-fast bacilli (AFB) smear, culture and nucleic acid amplification testing is generally lower from extrapulmonary specimen compared to sputum.⁴ 

Overall sensitivities of Xpert MTB/RIF assay were 88% and 98% among HIV infected and non-infected patients, respectively. In extrapulmonary specimen, a 2014 meta-analysis reported Xpert MTB/RIF median sensitivity varied by specimen type, with yield from lymph nodes (96%), CSF (85%) and gastric aspirates (78%) and pleural fluid (34%) and other serous fluid (67%).¹⁰ In this study, characteristic clinical and laboratory profiles of TBM in HIV-infected patients, adult patients of three specialist hospitals were evaluated to provide more insight into the empiric treatment of HIV-associated TBM.. 

In the present study, among 279 HIV patients with clinically-diagnosed meningitis, 24(8.6%) were CSF M. tuberculosis culture-positive. There was no CSF-AFB smear microscopy positive in any all culture-positive cases. A study conducted by Mon Mon on HIV- associated TBM meningitis patient attending Waibagi Specialist Hospital showed that the proportion of culture-positive cases was 13 cases (12.6%) out of 103 clinically-suspected meningitis patients and also reported that there was no CSF-AFB smear positive.¹¹ Although CSF smear is the primary mode of TB detection in many resource constrained settings, a number of smear-negative individuals often remain undiagnosed or receive delayed anti-TB therapy. CSF smear microscopy is low-cost and fairly easy to do, but it has very low sensitivity and also requires trained staff. These findings emphasize the danger that the diagnosis of TB may be missed or delayed in patients with HIV and more intensive use of CSF smear may not provide the solution. 

Although culture of CSF for M. tuberculosis is the diagnostic ‘gold standard’ for tuberculous meningitis, it takes too long for early diagnosis and treatment. The factors that influence sensitivity are similar to that of direct smear and microscopy. In most circumstances, the sensitivity and specificity of culture exceeds that of direct smear. The present study also showed sensitivity of culture was better than that of microscopy. However, once treatment has been started the sensitivity of culture falls quickly. Thwaites et al., stated only 5-10% of culture positive patients before treatment will have still positive cultures after 72 hours of four anti-tuberculosis drugs.¹² 

In the present study, most of the patients were currently treated with anti-TB drugs. That finding was also contributed to the factors causing lower culture positivity rate. Several studies have reported successful use of the Xpert MTB/RIF assay on extra- pulmonary samples, with overall sensitivities of over 80% and specificity reaching 100%.4 Xpert MTB/RIF assay could detect 15 cases (76.47%) out of 24 culture positive. Nine cases (23.53%) showed false negative in Xpert MTB/RIF. One study conducted on determinants of PCR (Xpert MTB/RIF) performance for TB diagnosis using specimens from different body compartments described that any contamination of specimen such as sugar, proteins or cellular debris commonly found in non-sputum specimens enriched after specimen processing (e.g. after centrifugation) could interfere with the amplification enzyme and thereby inhibit the PCR, leading to inaccurate or unreliable results, influencing the sensitivity of Xpert/MTB result.¹³

However, there was still limited information regarding how the performance of MTB/RIF is influenced by constituents of extra pulmonary specimens or any associated clinical factors. The study conducted by Bahr et al., reported that CSF centrifugation could increase sensitivity of Xpert MTB/RIF (72% sensitivity) when compared with uncentrifuged CSF (28% sensitivity).¹⁴ Nevertheless, the paucibacillary nature of TBM is a primary reason that microbiological confirmation remains challenging.

In the present study, there were 11 cases with culture negative showed positive in Xpert MTB/RIF. Similar discordant results were also seen in several previous studies. WHO stated that the specificity of false-positive results of Xpert MTB/RIF are likely to be linked to the detection of dead M. tuberculosis bacilli that would not be detected by culture.⁴

Regarding LAM /LFA antigen test for TBM cases, 2010 an autopsy study showed that the sensitivity of the LFA in unprepared CSF was 68% (95% CI: 47 to 84) for definite TBM and the specificity was 78% (95% CI: 66 to 87).⁸ Another study of LAM antigen assay for diagnosis of TBM in a high HIV setting, Africa, showed sensitivity of LAM antigen testing (95% CI:8; to 23), specificity 94% (95% CI: 85, 99), PPV 81% (95% CI: 54; 96), NPV 39% (95% CI: 31; to 48) and agreement 44% (95% CI: 36; to 52). When these LAM data were combined with the clinical index, the sensitivity and agreement improved significantly to 38% (95% CI: 28; to 49, p=0.0001) and 58% (95% CI: 50; to 66, p=0.01), respectively.¹⁵

In this study, LAM LFA antigen test Sensitivity, specificity, positive predicative value and negative predicative value were 42.86%, 95.49%, 57.69% and 92.09%, respectively. There are low sensitivity and fair specificity compared to other studies. The results from the present study can provide the diagnostic tool as clinical parameters and LAM-LFA test positivity to bacteriologically confirmed tuberculous meningitis among HIV patients and also may contribute to National TB Program for the consideration of diagnostic strategies for HIV-associated tuberculous meningitis patients.

Conclusion

This study detected the proportion of TBM cases among HIV-infected patients in three specialist hospitals in Yangon and also determines the non-specific clinical manifestations and high mortality in severe immunodeficiency patients. This study also indicated LAM LFA antigen test for CSF as a diagnostic tool which had low sensitivity with high specificity in bacteriologically confirmed TBM.

 

The authors declare that they have no competing interests.

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