Detecting tuberculosis in pregnant and postpartum women in Eswatini

Background Tuberculosis diagnosis in pregnancy is complex because tuberculosis symptoms are often masked by physiological symptoms of pregnancy. Untreated tuberculosis in pregnant and postpartum women may lead to maternal morbidity and low birth weight. Tuberculosis in HIV-positive pregnant women increases the risk of maternal and infant mortality. Objective This study aimed to determine tuberculosis prevalence stratified by HIV status and identify screening algorithms that maximise detection of active tuberculosis among pregnant and postpartum women in Eswatini. Methods Women were enrolled at antenatal and postnatal clinics in Eswatini for tuberculosis screening and diagnostic investigations from 01 April to 30 November 2015 in a cross-sectional study. Sputum samples were collected from all participants for tuberculosis diagnostic tests (smear microscopy, GeneXpert, MGIT culture). Blood and urine samples were collected from HIV-positive women for cluster-of-differentiation-4 cell count, interferon gamma release assay and tuberculosis lateral flow urine lipoarabinomannan tests. Results We enrolled 990 women; 52% were pregnant and 47% were HIV-positive. The prevalence of tuberculosis among HIV-positive pregnant women was 5% (95% confidence interval [CI]: 2–7) and among postpartum women it was 1% (95%CI: -1–3). Tuberculosis prevalence was 2% (95%CI: 0–3) in HIV-negative pregnant women and 1% (95%CI: -1–2) in HIV-negative postpartum women. The national tuberculosis symptom screening tool failed to identify women who tested tuberculosis-culture positive. Conclusion Routine tuberculosis symptom screening alone is insufficient to rule out tuberculosis in pregnant and postpartum women. Only sputum culture maximised the detection of tuberculosis, indicating a need to balance access and cost in developing countries.


Introduction
There has been a major decline in maternal mortality over the past two decades. 1 However, pregnancy poses several challenges to tuberculosis management because of its adverse effects on diagnosis and treatment outcomes. 2,3,4 The exact burden of tuberculosis among pregnant women is undefined. 5 The incidence of tuberculosis among postpartum women is unknown and difficulties with diagnosis suggest underestimation. 6 In 2015, the World Health Organization (WHO) advocated for research on new diagnostic methods targeting pregnant and postpartum women including HIV-positive women 7 and inexpensive tuberculosis screening algorithms for this population. 8 The clinical presentation of tuberculosis may be similar to some manifestations of pregnancy, making tuberculosis diagnosis in this population difficult. 6 The presence of tuberculosis during pregnancy may result in a threefold increase in adverse birth outcomes such as preterm birth, low weight at birth and foetal growth restriction. 8,9 Screening for tuberculosis in women of reproductive age is imperative, because concomitant tuberculosis disease causes higher case fatality rates than in men of the same age. 10 Active tuberculosis disease, especially when treated late or left untreated, is likely to result in severe adverse outcomes affecting both mother and baby, 11,12,13 with an estimated 3.4-fold increase in infant mortality. 14,15 The WHO has recommended three options for tuberculosis symptom screening based on availability of Xpert MTB/RIF assay, chest X-ray and also HIV status of the individual. 16 The option one algorithm uses a cough lasting more than 2 weeks to screen positive, http://www.ajlmonline.org Open Access option two uses any tuberculosis symptom and option three relies on positive chest X-ray findings. 16 Option two of the WHO symptom-screening algorithm gives a positive screen if a cough of any duration or any other tuberculosis symptom is evaluated. 16 The national tuberculosis symptom screening tool (NTBSS) adapted option one of the WHO screening algorithm, through which individuals screen positive if there is a cough of at least 2 weeks duration, 16 or a cough plus any other symptom of fever or unexplained loss of weight or night sweats, or if any two symptoms are present. 16,17 Despite developments in screening and diagnosis, emerging data show that the WHO-recommended four-symptom screen may miss persons with tuberculosis disease. 4 Studies on HIVpositive pregnant women found that the sensitivity of any one of the four tuberculosis symptoms was 28% in South Africa 18 and 42.9% in Kenya. 19 Few studies have considered the potential use of tuberculosis lateral flow urine lipoarabinomannan (LF-LAM) as an add-on to the tuberculosis screening algorithm for HIV-positive pregnant and postpartum women. 20 Xpert ® MTB/RIF testing has been rolled out in Eswatini and is the preferred tuberculosis diagnostic method for women. However, a study among HIVpositive pregnant women in Kenya reported Xpert ® MTB/RIF sensitivity of 43% and a specificity of 100%, when compared to Mycobacterium tuberculosis culture results 19 as the gold standard. Bactec MGIT 960 liquid culture has been shown to have a sensitivity and specificity of 100% and 93.3%. 21 Tests for latent tuberculosis infection have shown mixed value for determining the presence of the infection. 22 Existing tests for latent tuberculosis infection include the tuberculin skin test (TST) and the newer interferon-gamma release assays (IGRAs) but both have drawbacks. 23,24 The limitations of the TST include low sensitivity and specificity among HIVpositive patients and possibly among pregnant women. 11 Similarly, a study in Kenya using the T-SPOT TB IGRA showed a more than threefold increased risk of active tuberculosis or mortality among pregnant women who tested positive using IGRA. 25 In Eswatini, tuberculosis prevalence among pregnant women is not documented. Use of the recommended WHO foursymptom screen has identified very few pregnant women with positive results. 26 The objectives of this study were to determine the prevalence of bacteriologically confirmed tuberculosis among the study population of HIV-positive and HIV-negative pregnant and postpartum women and to identify effective tuberculosis screening algorithms.

Inclusion criteria
Participants were pregnant and postpartum women who were not on anti-tuberculosis treatment at enrolment or who had not taken anti-tuberculosis medicines, including isoniazid for tuberculosis preventive therapy, within the 2 months preceding enrolment, based on documented evidence from patient clinical records, and who provided informed consent. Four groups of women were enrolled: HIV-positive pregnant, HIV-negative pregnant, HIVpositive postpartum, and HIV-negative postpartum.

Study population and sample size
A sample size of 183 in each group was determined and a full narrative of sample size calculation is fully described in our protocol paper titled 'Screening in Maternity to Ascertain Tuberculosis Status (SMATS) study'. 4 Participants were consecutively enrolled until the sample size was reached.

Symptom screening
All participants were screened using the WHO-recommended national tuberculosis four-symptom screening (standard NTBSS) tool. Participants held clinic cards which were checked for evidence of tuberculosis symptom screening at their last clinical encounter to ascertain routine tuberculosis screening coverage at their previous visit. Participants were screened as positive using the standard NTBSS tool, if they had a cough lasting at least 2 weeks, 16 or a cough lasting less than 2 weeks plus any other symptom of fever or unexplained loss of weight or night sweats, or if any two symptoms were present. 16,17 Enhancements of the tuberculosis screening tool were done by adding to the four symptom screening tool any history of contact with a person on tuberculosis treatment or who had been diagnosed with tuberculosis and the presence of tuberculosis symptoms within the household inhabitants. We measured sensitivity, specificity and predictive values (positive and negative) of the WHO-recommended four symptom tuberculosis screening tool among HIV-positive and HIV-negative pregnant and postpartum women compared with sputum culture, the gold standard for M. tuberculosis detection.

Radiological procedures
Even though chest radiographs are not contraindicated in pregnancy, chest radiographs were only carried out among postpartum women (both HIV-positive and HIV-negative) to eliminate risk of radiation exposure to the foetus.

Specimen collection and laboratory procedures
Two samples of sputum (for Xpert ® MTB/RIF, smear microscopy and culture using BACTEC TM MGIT 960) were collected from all participants. 4 Sputum samples were collected through the production of spontaneous self-expectorated phlegm (preferred method), sputum induction through nebulising with hypertonic saline or, if both the above failed, the participant received a sputum container to take home and attempt to produce an early morning sputum sample and bring it back to the health facility. Testing of sputum samples was done at the National Tuberculosis Reference Laboratory in Mbabane. Tuberculosis culture was the gold standard test and in situations where sputum samples were insufficient for the three tests, culture was prioritised ahead of Xpert ® MTB/RIF and smear microscopy. A urine sample for the LF-LAM test and two 4 mL blood samples for IGRA testing and cluster-ofdifferentiation-4 (CD4) cell count testing were collected. Interferon-gamma release assays and LF-LAM were only done for HIV-positive women due to limited evidence on IGRA use 28 and existing WHO recommendations on LF-LAM use in HIV-positive individuals. 29 TST was also done and the induration was read after 48 hours -72 hours. IGRA and TST procedures were explained to all participants and only HIVpositive participants were then asked which test they preferred between IGRA and TST.
Sample collection and storage followed national standard operating procedures for urine and blood collection and manufacturer's instructions for the Determine TM Tuberculosis LF-LAM test (Abbott Laboratories, Lake Bluff, Illinois, United States) and IGRA testing. 4 All sputum specimens were analysed by means of: (1)

Data management and data analysis Data collection
The data collection tools were matched to the tools used for routine data collection at health facilities. Demographic fields in the data collection forms were adapted from client cards. Data fields for tuberculosis symptom screening were adapted from the national tuberculosis screening tool. Patient information was anonymised.

Data analysis
Data were entered in Epi Info™ (Centers for Disease Control and Prevention, Atlanta, Georgia, United States) and Research Electronic Data Capture (REDCap; Vanderbilt University, Nashville, Tennessee, United States), and data extraction tools were cross-checked to validate conflicting fields. Laboratory results were compared with the electronic study results file generated from the laboratory, and participant identity numbers were used to relate the data.
We evaluated both option one and option two of the WHO four symptom screening algorithms. TST numeric readings were recoded as positive, if the length of the induration was ≥ 5 mm for HIV-positive participants or ≥ 10 mm, if the participant was HIV-negative. 32,33 All other lengths, including 0 mm, were recoded as negative according to existing literature and CDC guidance on interpretation of TST results. 32,33 IGRA was done at a private laboratory according to manufacturer's recommendations and the differences in readings between QuantiFERON-tuberculosis Gold in-Tube tuberculosis antigen, tuberculosis nil and tuberculosis mitogen were used to interpret positive, negative and indeterminate results, respectively ( Figure 1 Frequencies and proportions were used to describe participant characteristics and related clinical data. Diagnostic parameters of sensitivity, specificity and positive and negative predictive values analyses for tuberculosis symptoms, and tuberculosis diagnostic tests were calculated in STATA version 13 (© 1985-2013 StataCorp LLC, College Station, Texas, United States). Using logistic regression, associations between culturepositive tuberculosis and HIV status and pregnancy or postpartum status variables were determined. Other sociodemographic and clinical factors were considered for inclusion in the multivariate model if the p-value was ≤ 0.1 on bivariate analysis. Factors that perfectly predicted the outcome were excluded. Estimates were reported with 95% confidence intervals (CIs) and corresponding p-values.

Performance of tuberculosis screening and diagnostic tests
The standard NTBSS tool failed to identify women with tuberculosis disease with a sensitivity of 0% (95% CI: 0-29) among HIV-positive and 0% (90% CI: 0-60) among HIVnegative participants ( Table 3). The enhanced screening tool, by including history of contact with a person with tuberculosis or presence of tuberculosis symptoms within the household of the participant, did not improve the sensitivity of the standard NTBSS tool. Only the inclusion of a tuberculosis contact history significantly improved the sensitivity of the algorithm to 18% among HIV-positive women, although specificity decreased from 94% to 62%.
M. tuberculosis was detected in two (1%) of the 361 Xpert MTB/RIF results, and no rifampicin resistance was detected in either one. No M. tuberculosis was detected in the remaining 359 (99%) samples. Sputum smear microscopy was done on 724 (73%) participants, and 4 (1%) had acidalcohol-fast bacilli. LF-LAM was done on 411 (87%) participants and 327 (80%) had culture results. When compared to culture, the sensitivity of both the tuberculosis screening and the tuberculosis diagnostic tests were less than 50% in both HIV-positive and HIV-negative women ( Table 3).

Association between socio-demographic and clinical covariates and tuberculosis culture diagnostic algorithm
Specific algorithms could not be analysed due to the missing data of tests used to construct the different algorithms, as well as low prevalence of tuberculosis symptoms among our   study participants. The study showed that those who were HIV-positive had a threefold risk of culture-positive tuberculosis compared to HIV-negative individuals (odds ratio = 3.23; 95% CI: 1.00-10.40, p = 0.05) ( Table 4). Those residing in the Central region (odds ratio = 0.08; 95% CI: 0.01-0.61, p = 0.015) and Southern region (odds radio = 0.19; 95% CI: 0.04-0.89, p = 0.035) were independently less likely to have culture-positive tuberculosis compared to the northern region (reference region) and (Table 4).

Summary of key findings
According to our review, this is a unique study in this setting to determine the burden of tuberculosis among pregnant and postpartum women regardless of the presence of tuberculosis symptoms. We observed that higher proportions of pregnant women (84%) were previously screened for tuberculosis during their last clinic visit prior to enrolment compared to postpartum women (60%). However, these were lower than the universal screening (99%) reported among people living with HIV attending antiretroviral therapy clinics in Eswatini. 35 Even though 80% of participants who were confirmed to have tuberculosis disease were pregnant, there were no statistical differences between the prevalence of tuberculosis in pregnant and postpartum women. The highest prevalence of tuberculosis was among HIV-positive pregnant women (5%), which is comparable to the 3.3% observed in neighbouring South Africa. 11 Although 93% of participants who were found to have active tuberculosis reported no tuberculosis symptoms, there were no differences in tuberculosis prevalence between those reporting symptoms and those with no symptoms. A study conducted in South Africa found a higher tuberculosis prevalence among patients who did not report symptoms of tuberculosis. 18 A study from Ethiopia did not find any person with active tuberculosis disease among pregnant women but did not test for tuberculosis among those who did not have tuberculosis symptoms. 36 This has significant public health implications for tuberculosis control, considering previous reports that asymptomatic patients with culture-positive tuberculosis can transmit tuberculosis. 18 The WHO four-symptom NTBSS screening tool failed to identify women with active tuberculosis disease, as the majority of women with confirmed tuberculosis disease did not have symptoms of tuberculosis. Almost a third of participants who had a TST done did not have results, because participants did not come back within the stipulated time for reading, despite the provision of transport imbursements for additional visits for TST reading.
Pregnancy is known to suppress the T1-helper proinflammatory response, resulting in masking of tuberculosis symptoms and increased susceptibility to M. tuberculosis reactivation 8 and primary infection with M. tuberculosis. 37 Women are unlikely to show typical symptoms like sweating at night and fever, and these are further masked by pregnancy. 8,17 Weight loss can be masked by physiological changes during pregnancy. 18 The low sensitivity of the tuberculosis screening tool has been reported in many other studies. 18,19,38,39 Adapting the screening tool to include a history of tuberculosis contact as an independent indicator of positive tuberculosis screen improved sensitivity by 18% but only in HIV-positive women. This sensitivity (18%) is still too low for effective tuberculosis case finding and ruling out tuberculosis among pregnant and postpartum women. LaCourse et al. 19 also showed poor performance of the foursymptom screening tool in HIV-positive pregnant women in Kenya. Tuberculosis diagnosis in pregnancy is often delayed due to atypical symptoms. 11,39,40 Culture prevailed as the reliable gold standard to diagnose tuberculosis; some authors recommend that it should be mandatory for establishing tuberculosis diagnosis in this group. 11,39 A false-negative symptom screening, which is the initial screening method for triaging for tuberculosis diagnostic testing, will often lead to a delay in diagnosis and treatment of active tuberculosis and consequently poor foetal and maternal outcomes. 17 A false-positive screening result leads to inconvenient and costly laboratory procedures. 17 However, the routine symptom screening tool had high negative predictive values (97% -99%), which supports its utility in identifying people who are unlikely to have tuberculosis, especially people living with HIV. Therefore, those who screen negative with the standard NTBSS tool and are at high risk of progressing from latent tuberculosis to active tuberculosis can be given tuberculosis preventive therapy in this setting of high HIV-tuberculosis burden. 39,41 We demonstrated that TST had poor performance when used as a screening method for exposure to tuberculosis and its feasibility is further challenged by a third of participants who were lost to follow-up for a reading of skin reaction. However, alternative follow-up methods, including home visits, should be included to complete the TST readings. IGRA was a less preferred screening method compared to TST, possibly due to the need for blood draw. In addition, IGRA demands laboratory infrastructure 42 compared to TST and is currently available only in private laboratories in this setting.

Strengths and limitations
We considered it a strength that our methodology included participants who did not have the usual symptoms of tuberculosis, minimising the risk of under-reporting of true tuberculosis prevalence in this study population. 39,43 We also induced sputum in women unable to produce sputum spontaneously, thus maximising the tuberculosis diagnostic yield. 19 In addition to symptom screening, we attempted five tuberculosis tests for all participants.
We had planned to test 10 different algorithms using different testing combinations and ordering of individual diagnostic tests. However, given the low agreement, the low number of positives, and the poor performance of the WHO symptom screening tool (several of the algorithms began with the WHO symptom screening), the analysis was of no utility, and we did not include these results in this report. Although unintended by the study design, more than half of the participants did not have an Xpert ® MTB/RIF assay done, which is a near point-of-care test that allows results to be quickly available (as early as 2 h) to clients and service providers, leading to a quick clinical management decision, unlike culture which may take several weeks.

Conclusion
The four-symptom screening tool appears likely to miss women with active tuberculosis. Without sensitive, symptom-based tuberculosis screening in this subpopulation, a high index of suspicion of tuberculosis is necessary and factors such as a history of tuberculosis contact should prompt clinicians to consider tuberculosis in their differential diagnosis, especially in a setting of high HIVtuberculosis burden. Bold, deliberate decisions to invest in laboratory-based, quality-assured culture testing are required to maximise detection of people who have active tuberculosis disease in countries with a high HIVtuberculosis burden in order to end tuberculosis by 2035 as envisaged by the WHO's End TB Strategy. 44 However, the feasibility of increasing access to culture in this setting is confronted by costs related to culture testing, transportation, specimen storage and lengthy waiting periods for culture results by clinicians and patients. Therefore, low-income and middle-income countries should strike the right balance to ensure access to culture for those who could benefit from culture and availability of the newer, more sensitive Xpert ® MTB/RIF Ultra platform for rapid tuberculosis diagnosis.
Prevalence of tuberculosis was particularly high (4.5%) among HIV-positive pregnant women and low (0.6%) among HIV-negative postpartum women. Although we were unable to test different tuberculosis screening algorithms, the poor performance of the standard NTBSS tool that serves as the entry to tuberculosis services highlights the challenge of diagnosing tuberculosis in pregnant and postpartum women.

Data availability statement
Data sharing is not applicable to this article as no new data were created or analysed in this study.

Disclaimer
The views expressed in this article are views of the authors and not an official position of the authors' institutions or funder.