key: cord-0316817-u4c0gvti
authors: Byanyima, P.; Kaswabuli, S.; Musisi, E.; Nabakiibi, C.; Zawedde, J.; Sanyu, I.; Ssesolo, A.; Andama, A.; Worodria, W.; Huang, L.; Davis, J. L.
title: Feasibility and Sensitivity of Saliva GeneXpert MTB/RIF Ultra for Tuberculosis Diagnosis in Adults in Uganda
date: 2022-03-18
journal: nan
DOI: 10.1101/2022.03.16.22272031
sha: 02e20d02028f8e8524c23cea770adc7a393ca55c
doc_id: 316817
cord_uid: u4c0gvti

The objective of this prospective, observational study carried out at China-Uganda Friendship Hospital-Naguru in Kampala, Uganda, was to determine the performance of GeneXpert MTB/RIF Ultra (Xpert) testing on saliva for active tuberculosis (TB) disease among consecutive adults undergoing diagnostic evaluation. We calculated sensitivity to determine the diagnostic performance in comparison to that of the composite reference standard of Mycobacterium tuberculosis liquid and solid cultures on two spot sputum specimens. GeneXpert Ultra on saliva had a sensitivity of 90% (95% confidence interval [CI], 81-96%); this was similar to that of sputum fluorescence smear microscopy (FM) of 87% (95% CI, 77-94%). Sensitivity was 24% lower (95% CI for difference 2-48%, p=0.003) among persons living with HIV (71%, 95%CI 44-90%) than among persons living without HIV (95%, 95%CI 86-99%) and 46% lower (95% CI for difference 14-77%, p<0.0001) among sputum microscopy positive (96%, 95% CI 87-99%) than among sputum microscopy negative patients (50%, 95% CI 19-81%). Semi-quantitative Xpert grade was higher in sputum than in paired saliva samples from the same patient. In conclusion, saliva specimens appear to be feasible and similarly sensitive to sputum for active TB diagnosis using molecular testing, suggesting promise as a non-sputum diagnostic test for active TB in high-burden settings.

INTRODUCTION 1 ver the last quarter century, improvements in diagnosis and treatment of people with 2 tuberculosis (TB) have gradually reduced mortality, but large gaps in detection and 3 treatment persist that contribute to substantial ongoing morbidity and mortality [1] . Among 4 several available strategies to facilitate rapid, same-day diagnosis of TB, testing sputum with the 5 GeneXpert MTB/RIF Ultra molecular assay [2, 3] is the most sensitive and most readily 6 available approach. Unfortunately, there are several operational challenges associated with 7 collecting sputum for diagnosis of pulmonary TB. First, coughing during sputum expectoration 8 or sputum induction generates aerosols that may facilitate TB transmission [4] . Second, some 9

individuals are unable to produce sputum, including young children, those with dry cough, and 10 the severely ill/severely debilitated. Given these limitations of sputum for TB diagnosis, in 2014 11 the World Health Organization (WHO) issued guidelines for developers of a future non-sputum 12 test for active TB diagnosis, including a target product profile suggesting that it should have a 13 minimum diagnostic accuracy similar to sputum GeneXpert MTB/RIF on sputum smear-negative 14 individuals (i.e., sensitivity ≥ 68%, specificity ≥ 98%) [5] . 15 16 One alternative sample type with great promise for diagnosis of pulmonary TB is saliva, which is 17 easy to collect, with minimal risk of generating aerosols. Although Stop TB Partnership 18 guidelines discourage collection of salivary sputum samples because they have lower diagnostic 19 yield for acid-fast bacilli (AFB) by microscopy or culture, the diagnostic yield of TB molecular 20 testing appears to be more promising. In a previous study of 1782 smear-negative adults 21 undergoing evaluation for active TB, for example, we found that salivary sputum provided a 22

substantially higher diagnostic yield and sensitivity for culture-positive TB than other sputum 23 O . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted March 18, 2022. ;  https://doi.org/10.1101/2022.03.16.22272031 doi: medRxiv preprint types, implying incremental value to using oral samples at least as a supplement to sputum [6] . 24

Using a different sampling technique, oral swabs, Wood and colleagues showed that oral nylon 25 swabs repeatedly tested positive for TB via IS6110 polymerase chain reaction testing in 90% of 26 South African patients with TB confirmed by sputum GeneXpert MTB/RIF testing, suggesting 27 that TB is present in the oral cavity [7] . A subsequent study of 50 adults with possible TB in 28

Uganda found similar sensitivity of 88%, albeit with lower specificity. Saliva is also now widely 29 used for molecular diagnosis of COVID-19, where it has high sensitivity, even among patients 30 without symptoms [8] . Using saliva as a diagnostic specimen in the COVID-19 context has been 31 shown to reduce aerosol exposure for health workers and eliminate the need for personal 32 protective equipment because it is self-collected [9] . This raises the possibility that saliva alone 33 could be used as a TB diagnostic when paired with next generation and ultra-sensitive molecular 34 Hospital-Naguru in Kampala, Uganda; patients with a prior history of TB within the past two 46 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. performed GeneXpert MTB/RIF testing on the remainder [13] . Finally, staff sent a third sputum 59 sample for mycobacterial culture on solid media and liquid culture.. All cultures were performed 60 at the Makerere University Mycobacteriology Laboratory, and staff performing the cultures were 61 not provided with clinical information about the study participants. At least two hours after 62 sputum collection, the patients were asked to submit at least 1 mL of saliva placed into a sterile 63 specimen cup for GeneXpert MTB/RIF testing; all participants were instructed not to cough prior 64 to saliva collection. Saliva specimens were processed for GeneXpert MTB/RIF using a sample 65 reagent to saliva volume ratio of 1:1, and all other steps followed the manufacturer's 66 recommendations for extra-pulmonary body fluid specimens [13] . Sputum was collected prior to 67 TB treatment initiation, and saliva was collected prior to or within two hours of TB treatment 68

initiation. Finally, all participants without a prior known HIV diagnosis were offered HIV testing 69 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 18, 2022 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. and 10 (12%) had 1-9 AFB seen per 100 high-powered fields. 13 (16%) were AFB smear-110 negative and one (1%) was missing an AFB smear microscopy result (Table 1) We also compared the semi-quantitative results of bacilli by GeneXpert for both saliva and 125 sputum, as shown in Table 2 In a prospective, observational study of consecutive sputum GeneXpert-positive TB patients in a 135 high-burden setting, we showed that diagnosis of TB using GeneXpert Ultra on saliva is feasible 136 and had a high sensitivity relative to a rigorously defined reference standard. This finding has 137 significant implications for the diagnosis of TB and potentially also for TB infection control. 138 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101/2022.03.16.22272031 doi: medRxiv preprint Using sputum specimens for TB diagnosis poses a number of challenges, since some individuals 139 such as those with non-productive cough and young children find expectoration challenging, and 140 the associated generation of sputum aerosols poses an infection control risk for health care 141 workers and nearby patients [14] . The development of novel testing strategies that employ non-142 sputum samples for TB has been identified as a priority by the WHO, and the sensitivity 143 measured in our study is consistent with WHO's minimum target-product profile for a non-144 sputum-based test, with similar sensitivity to sputum GeneXpert among a population of 145 predominantly sputum microscopy-positive and HIV-negative individuals. Although our 146 alternative strategy of salivary GeneXpert exceeds WHO's optimal targets for cost ($4) and turn-147 around time (20 minutes) for a non-sputum-based test, if GeneXpert on saliva were shown to 148 perform well in populations for whom sputum collection is less feasible for the reasons described 149 above, the willingness to pay for and wait for results might be higher. 150

The use of saliva for molecular diagnosis of TB was first described in a convenience sample of 152 52 adult TB patients in Japan who were evaluated using a lab-developed, nested PCR assay that 153 was shown to have a sensitivity of 98% [15] . A more recent study of 44 sputum smear-and 154 culture-positive TB patients, including 35 in South Africa and 9 in South Korea, reported on 155 saliva as having a very low sensitivity of 39% for TB testing [16] . Sputum mycobacterial load 156 was similarly high (100% smear-positive in the South Africa/South Korea study vs. 87% in our 157 study), so these differences in diagnostic performance might be attributable to differences in 158 either sample collection or specimen processing. For example, participants were instructed to 159 rinse their mouths prior to specimen collection in the South Africa/South Korea study but not in 160 our study. Second, the South Africa/South Korea study diluted one part of the sample in two 161 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 1 0 parts of sample reagent as recommended by the manufacturer for sputum, while we used a 1:1 162 dilution ratio as recommended for cerebrospinal fluid, another extra-pulmonary specimen 163 without a mucoid matrix [17] . Finally, we used the GeneXpert MTB/RIF Ultra cartridge, which 164 has ten-fold better analytic sensitivity than the earlier generation GeneXpert MTB/RIF cartridge. 165

To our knowledge, we are among the first to report the performance of GeneXpert MTB/RIF 166 Ultra on saliva. 167 168 Previous studies have examined the sensitivity of a variety or oral specimens for diagnosis of 169 TB. We previously showed that oropharyngeal wash specimens paired with a lab-developed PCR 170 assay had a high sensitivity for TB diagnosis in reference to sputum mycobacterial culture on 171 previously frozen and thawed sputum, but a subsequent study failed to confirm these results [18, 172 19] . A study of Mtb PCR on buccal swabs of South African TB patients and US controls showed 173 high sensitivity (90%) and specificity (100%), although the case-control design may have 174 inflated diagnostic accuracy [7] . A recent study from the US was among the first to show that 175 saliva is a viable and accurate specimen for diagnosis of SARS-CoV2, and more sensitive and 176 less variable than nasopharyngeal swab specimens [20] . Another study carried out in Thailand 177 using saliva for diagnosis of SARS-CoV2 showed similar results, with saliva providing a 178 sensitivity of 84% and a specificity of 99% [21] . Collectively, these studies suggest that saliva is 179 a very promising novel specimen for diagnosis of respiratory tract infections. 180 181 There were a few limitations to our study. First, because the primary study objective was to 182 evaluate feasibility and preliminary sensitivity, we did not include patients with non-productive 183 cough or children, two ideal target populations for salivary testing. If, as seems plausible, these 184 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 1 1 populations have more paucibacillary disease, diagnostic sensitivity could be lower in these 185 populations, as suggested by the lower sensitivity observed among sputum smear-negative 186 individuals and persons living with HIV. However, in the current study, we found that even 187 though saliva is more paucibacillary than sputum as assessed by GeneXpert's semi-quantitative 188 measurement of mycobacterial load, diagnostic sensitivity was similar between the two specimen 189 types, likely because of the extremely low threshold of analytic sensitivity of the GeneXpert 190 Ultra assay [22] . Secondly, to conserve costs in this preliminary study, we did not enroll non-TB 191 patients to serve as controls, a choice that prevented us from estimating diagnostic specificity. 192

However, a recent systematic review found that both GeneXpert MTB/RIF assays have a high 193 specificity on a variety of body fluid types [23] . Thirdly, our sample size was small, especially 194 for persons living with HIV and for sputum smear-negative patients, which limited our ability to 195 develop precise accuracy estimates for these and other subgroups. including swabs, would also be useful. Finally, studies evaluating the relative impacts of salivary 206 versus sputum testing on infection control proxies and/or on outcomes would also be valuable. 207 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 

Very Legend: Shading intensity is proportional to the frequency of paired results by semi-quantitative grade across the two sample types.

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Xpert MTB/RIF and Xpert MTB/RIF Ultra for pulmonary tuberculosis and rifampicin resistance in adults

Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis. The Lancet Global Health

Cough aerosols of Mycobacterium tuberculosis predict new infection. A household contact study. American journal of respiratory and critical care medicine

for New Tuberculosis Diagnostics: Report of a Consensus Meeting

Sputum quality and diagnostic performance of GeneXpert MTB/RIF among smear-negative adults with presumed tuberculosis in Uganda

Detection of Mycobacterium tuberculosis DNA on the oral mucosa of tuberculosis patients. Scientific reports

Saliva or nasopharyngeal swab specimens for detection of SARS-CoV-2

Saliva Testing Is Accurate for Early-Stage and Presymptomatic

Rapid Molecular Detection of Extrapulmonary Tuberculosis by the Automated GeneXpert MTB/RIF System

Suitability of saliva for Tuberculosis diagnosis: comparing with serum. BMC infectious diseases

PCR method is essential for detecting Mycobacterium tuberculosis in oral cavity samples. Oral microbiology and immunology

Exploring Alternative Biomaterials for Diagnosis of Pulmonary Tuberculosis in HIV-Negative Patients by Use of the GeneXpert MTB/RIF Assay

Xpert MTB/RIF implementation manual: technical and operational 'how-to'; practical considerations

Polymerase chain reaction of secA1 on sputum or oral wash samples for the diagnosis of pulmonary tuberculosis

Nucleic acid amplification tests for diagnosis of smear-negative TB in a high HIV-prevalence setting: a prospective cohort study

Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2

Saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: a cross-sectional study

The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Pointof-Care Testing

Xpert MTB/RIF Ultra and Xpert MTB/RIF assays for extrapulmonary