Signature: DATE:. Copyright Act 1999 and international and national enactment, in that behalf on

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1 Evaluation of Tuberculosis Score Charts against Bacteriologically Confirmed Pulmonary Tuberculosis by Induced Sputum among Children at Bugando Medical Centre Issa Sabi, MD A dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Medicine (Paediatrics and Child Health) of the Catholic University of Health and Allied Sciences

2 DECLARATION AND COPYRIGHT I Issa Sabi hereby declare that the work presented in this dissertation has not been presented and will not be presented to any other university for similar or any other degree award. Signature: DATE:. This dissertation is a copyright material protected under the Berne Convention, the Copyright Act 1999 and international and national enactment, in that behalf on intellectual property. It may not be reproduced by any means, in full or in part, except for short extracts in fair dealing, for research or private study, critical scholarly review or discourse with an acknowledgement, without written permission of the directorate of postgraduate studies, on behalf of both the author and the CUHAS- Bugando. i

3 CERTIFICATION The undersigned certify that they have read and hereby recommend acceptance for examination by the Catholic University of Health and Allied Sciences-Bugando, a dissertation titled: Evaluation of Tuberculosis Score Charts Against Bacteriologically Confirmed Pulmonary Tuberculosis by Induced Sputum among Children at Bugando Medical Centre in partial fulfilment for the requirement of the award of degree of Masters Medicine (Paediatrics and Child Health). Signature Date.. Dr. Rogatus Kabyemera, MD, M.MED Peaditrician, Department of Peadiatrics Bugando Medical Centre Signature Date.. Prof. Stephen.E. Mshana, MD, M.Med, PhD Associate Professor of Microbiology and Immunology Catholic University of Health and Allied Sciences - Bugando Signature Date.. Dr. Robert N. Peck, MD Assistant Professor of Medicine and Pediatrics, Weill Cornell Medical College Consultant physian, Department of Internal Medicine, Catholic University of Health and Allied Sciences - Bugando ii

4 DEDICATION This work is dedicated to my lovely wife Hadija iii

5 ACKNOWLEDGEMENT This work is the result of the contributions of many people. I would like to express my sincere gratitude to: My supervisors Dr. Rogatus Kabyemera, Prof. Stephen E. Mashana and Dr. Robert Peck, for their continuous support and valuable inputs to this work. The Department of Peadiatrics and Child Health, BMC for their valuable contributions and continuous support towards this study. Mrs Lisa E. Gerwing-Adima, TB laboratory of BMC for her valuable advice and continuous support in all laboratory related matters. Dr Kassanga, department of radiology of BMC, despite his busy schedule he was able to interpret chest X-rays of all patients enrolled in the study. Dr. Benson Kidenya, Department of Biochemistry of Catholic university of health and allied sciences, for statistical analysis. Dr Andrea Rachow, Department of Infectious Diseases & Tropical Medicine, Klinikum der Universitaet Muenchen and Dr. Petra Clowes, NIMR-Mbeya Medical Research Centre (NIMR-MMRC) for their great support and facilitating the availability of LJ culture slants and related consumables and for facilitating the training and providing some of the equipments for sputum induction. The office of the Regional TB and Leprosy Coordinator in Mwanza, for realizing the importance of this study and provide the support for Xpert MTB/RIF assay and its related consumables. iv

6 The parents and care takers of the all the children who were enrolled in the study for their consent to participate in this study. Without their consent this study would not have been done. My fellow residents, for their continuous support and inputs in this study. Lastly but not least my family for bearing with me during the time of this study. I spent very little time with them for the entire period of the study. v

7 Table of Content DECLARATION AND COPYRIGHT... i CERTIFICATION... ii DEDICATION... iii ACKNOWLEDGEMENT... iv LIST OF TABLES... xi LIST OF FIGURES... xii OPERATIONAL DEFINITION... xiii ABBREVIATIONS... xv ABSTRACT... xvii 1.0 INTRODUCTION Background Problem Statement Rationale of the Study Research Questions Objectives Broad Objective... 4 vi

8 1.5.2 Specific Objectives LITERATURE REVIEW Difficulties in Diagnosing Pulmonary TB in children Pediatric TB Score chart Specimen Recovery Methods Confirmation of Pulmonary TB in children MATERIALS AND METHODS Site Description Study design Sample size Study Population Data collection Standard diagnostic procedures Laboratory procedures Study duration and management of study subjects Outcome of the study Study Variables vii

9 3.8 Statistical analysis Data quality and assurance Ethical consideration RESULTS Baseline Characteristics Specimen recovery Specimen recovery Common presenting symptoms Radiological Characteristics Patients Follow-up Yield for Mycobaterium tuberculosis from one induced sputum The proportion of PTB cases Sensitivity, Specificity, & Positive/Negative predictive values Factors associated with microbiologically confirmed PTB DISCUSSION Sputum Induction as a method of specimen recovery method Proportion of patients who had pulmonary tuberculosis viii

10 5.3 Sensitivity and specificity of the TSCs Factors associated with confirmed TB CONCLUSIONS RECOMMENDATIONS LIMITATIONS OF THE STUDY REFERENCES APPENDICES Data Collection Form Standardized Chest X-ray Review Tool Information Sheet and Informed Consent Form Information Sheet and Informed Consent Form Karatari ya maelezo na form ya ridhaa Sputum induction Diagnostic Scoring Systems Keith Edward Score Chart MASA System Fourie Scoring System ix

11 Brazilian MoH Scoring System Tuberculin Skin Testing Study Budget Test for HIV Diagnosis Research Clearance Certificate x

12 LIST OF TABLES Table 1: Baseline characteristics of study participants Table 2: Follow up among patients enrolled in the study Table 3: Yield for MTB from one induced sputum among patients enrolled in the study Table 4: TB cases based by Peadiatrics TB score charts and Diagnostic classification among patients enrolled in the study Table 5: Sensitivity, specificity, PPV and NPV of TSCs against microbiologically confirmed pulmonary TB Table 6: Baseline characteristics associated with confirmed TB among patients enrolled in the study Table 7: Radiological features associated with confirmed Pulmonary TB among patients enrolled in the study xi

13 LIST OF FIGURES Figure 1: Study flow diagram Figure 2: Common presenting symptoms among patients enrolled in the study xii

14 OPERATIONAL DEFINITION Children were categorized by using classification group on the basis of different clinical, radiological, and microbiological information as shown below: Pulmonary TB: A child with pulmonary TB symptoms who had M. tuberculosis confirmed in sputum by culture or Xpert MTB/RIF assay, or a child without M. tuberculosis confirmation but had clinical and radiologic features that prompted empirical treatment for pulmonary TB. Pulmonary TB case was further categorized as shown below based on diagnostic classification as previously proposed by Graham et al (1). In addition to that classification Xpert MTR/RIF assay was also a confirmatory test for pulmonary tuberculosis in this study. Confirmed tuberculosis: Probable tuberculosis: Patients were classified as confirmed TB when they presented with: 1. At least 1 of the signs and symptoms suggestive of tuberculosis and 2. Microbiological confirmation is obtained by culture, Xpert MTB/RIF assay Patients were classified as probable tuberculosis cases when they presented with: 1. At least 1 of the signs and symptoms suggestive of tuberculosis and 2. Chest radiography is consistent with intrathoracic disease due to Mycobacterium tuberculosis and 3. There is at least 1 of the following: (a) A positive clinical response to anti-tuberculosis treatment (b) Documented exposure to M. tuberculosis (c) Immunological evidence of M. tuberculosis xiii

15 infection Possible tuberculosis: PTB unlikely: Not PTB tuberculosis: Patients were classified as possible tuberculosis when they presented with at least 1 of the signs and symptoms suggestive of tuberculosis and either (1) One of the following: (a) A positive clinical response to anti-tuberculosis treatment (b) Documented exposure to M. tuberculosis (c) Immunological evidence of M. tuberculosis infection or (2) Chest radiography is consistent with intrathoracic tuberculosis disease NB: if at least 1 of (1) and (2) are both present, then this case should be classified as probable tuberculosis. Symptomatic but not fitting the above definitions and no alternative diagnosis established Established alternative diagnosis or no clinical deterioration on follow-up in the absence of tuberculosis therapy xiv

16 ABBREVIATIONS AFB BAL BMC CUHAS CXR GL HIV IUATLD LIP Acid Fast Bacilli Bronchoalveolar Lavage Bugando Medical Centre Catholic University of Health and Allied Sciences Chest X-ray Gastric Lavage Human Immunodeficiency Virus International Union Against Tuberculosis and Lung Disease Lymphocytic Interstitial Pneumonia LJ Löwenstein - Jensen MASA MoHSW MTB NPA NTLP PCP PPTB PTB RIF Medical Association of South Africa Ministry of Health and Social Welfare Mycobacterium Tuberculosis Nasopharyngeal Aspiration National Tuberculosis and Leprosy Control Programme Pneumocystis jerovecii pneumonia Paediatric Pulmonary Tuberculosis Pulmonary Tuberculosis Rifampicin xv

17 TB TSC TST WHO Tuberculosis Tuberculosis Score Chart Tuberculin Skin Test World Health Organization xvi

18 ABSTRACT Background: Bacteriological confirmation of childhood TB continues to be difficult due to the unavailability of diagnostic facilities, difficulties in obtaining samples, and poor performance of smear microscopy. As a result various peadiatric TB scoring tools were invented in order to help in clinical diagnosis. Therefore this study was done to evaluate various peadiatric tuberculosis score charts for the diagnosis of pulmonary TB against bacteriologically confirmed pulmonary TB in the paediatric department of Bugando Medical Centre (BMC). Material and Methods: A cross sectional study was conducted from October 2013 to April 2014 among children suspected to have pulmonary TB at BMC. All children were assessed for TB by using pediatric tuberculosis score charts (Keith Edward, MASA, Fourie and Brazil MOH). Sputum induction was attempted in all patients to obtain sputum which was examined by fluorescent acid fast smear, LJ culture and Xpert MTB/RIF assay. Sensitivity, specificity, positive predictive values and negative predictive value of the score charts against pulmonary TB cases were determined. Results: A total of 192 patients were enrolled in the study over a period of 5 months. Males formed 53.7% of all the participants. Median (IQR) age was 1.9 ( ) years. Sputum specimen was obtained in 187 (97.4%) patients; sputum induction was successfully performed in 183 (95.8%) patients. Forty (20.8%) patients were diagnosed with pulmonary TB; among them 10 (5.2%) had confirmed pulmonary TB. Generally, the sensitivity of pediatric TB score charts was poor for diagnosing pulmonary TB (10% - 50%). No baseline characteristics had significant association with confirmed pulmonary TB. Airspace opacification and cavities on chest X-ray had strong association with confirmed PTB with p-values of <0.01 and 0.01 respectively. Conclusion: Sensitivity and specificity to detect pulmonary TB with peadiatric TB score charts evaluated in this study were very low. Induced sputum is possible in our setting therefore bacteriological confirmation of pulmonary TB should be attempted in every child suspected to have TB. CXR components should be incorporated in all TB scores to increase the sensitivity. xvii

19 1.0 INTRODUCTION 1.1 Background Tuberculosis (TB) is still a major public health problem and associated with high morbidity and mortality; globally about nine million new cases and two million deaths occur annually. It is estimated that about one-third of the world population has been infected by the mycobacterium that causes TB. Childhood TB contributes a significant proportion of the global TB burden. Of the estimated 9 million new TB cases that occur annually about one million cases (11%) occur in children less than 15 years of age. About 75% of the reported TB cases in children occur in 22 high TB burden countries (2,3). According to the Global TB report of 2011, Tanzania ranks at number 20 among 22 high TB burden counties. Data from the National TB and Leprosy Programme (NTLP) from 2004 to 2011 show that childhood TB contributes about 10% to all notified cases in Tanzania. In 2010 alone, there were 5,216 TB case notifications in children less than 15 years of age, which accounted for 8.7% of the total new case notifications. However, data from studies done in Kilimanjaro and Mwanza regions suggest that the contribution of childhood TB in Tanzania might be even higher than the estimated worldwide average (4,5). The magnitude of TB disease among children in Tanzania is difficult to ascertain due to challenges in diagnosing and reporting. In a retrospective review of the TB registry at Bugando Medical Centre (BMC) a total number of 1,023 TB patients were treated from January to December 2012 including 79 children of less than 15 years of age who contributed about 7% of the reported TB cases. There were no confirmed TB 1

20 cases; therefore all children were treated based on either Keith Edward score chart or X-ray findings. In most resource-constraint settings, the diagnosis of pulmonary TB (PTB) in children is usually based on history of TB contacts, positive Tuberculin Skin Test (TST) and radiological findings suggestive of TB. However, in developing countries the diagnosis of PTB is usually difficult because the contact history is often not clear, the tuberculin test is often false positive due to routine BCG immunization, and because of difficulties in interpreting chest radiographs especially in HIV-coinfected patients where HIV-related pulmonary disease such as lymphocytic interstitial pneumonitis (LIP). But also common viral and bacterial infections of the lung may present with similar symptoms and radiological patterns. Confirmation of tuberculosis by culture, which is considered the gold standard for confirmed TB diagnosis, is important where, for example, there is difficulty in clinical diagnosis, the contact history is not known or drug sensitivity is required. Sputum induction is the preferred method for collecting sputum in children because children do not have enough strength to expectorate. Several studies have shown that induced sputum has a higher diagnostic yield than expectorated sputum in children and is comparable to or better than gastric lavage specimens (6 10). Sputum induction has been shown to be a safe and effective procedure even in children as young as 1 month of age (8,11). Currently in Tanzania, it is recommended that sputum induction is performed at district, regional and zonal referral hospitals, where appropriate equipments and personnel are available (Tanzania National Guideline for Management of TB in 2

21 Children, 2012). Despite of availability of experts at BMC, sputum induction to obtain sputum sample is so far not performed on children suspected to have TB. 1.2 Problem Statement Accurate and consistent diagnosis is critical for the effective management of children with TB, for measuring the precise burden of childhood TB, the TB-related morbidity and mortality, and to establish drug susceptibility patterns. In our setting, despite of the availability of facilities and expertise bacteriological confirmation of childhood TB continues to be difficult due to the difficulty in obtaining samples in children, poor performance of smear microscopy, long turnaround time for culture results and the perception that microbiologic yield is low. Currently, several studies have confirmed that microbiologic confirmation is feasible and useful to exclude drug resistant TB (6). Clinical scoring systems such as the Keith Edward Scoring Chart (12) have commonly been used as a diagnostic tool to diagnose TB in children. However those systems are poorly validated, may not be generalizable to different settings, and are not adapted for use in HIV-infected children (6,13). 1.3 Rationale of the Study This study evaluated the Keith Edwards, MASA, Fourie and Brazilian MoH diagnostic score charts against culture for the diagnosis of peadiatric PTB. While in other setting, various clinical scores have been shown to have low sensitivity and low specificity especially when applied in HIV-endemic areas (14,15), these scoring systems are are still being used in our setting for want of a better alternative. 3

22 Sputum induction is not routinely done at BMC and many hospitals in Tanzania. Therefore, this study also investigated the feasibility of sputum induction as a method for sample collection, in order to improve the diagnosis of peadiatric PTB in our setting. This study was also aiming to provide baseline data from our local setting that would be helpful to plan future studies of childhood TB. Prior studies of pediatric TB at Bugando have not included confirmation of PTB with culture and microscopy, so the true incidence of TB among pediatric inpatients at BMC is not yet known. 1.4 Research Questions What is the sensitivity and specificity of pediatric tuberculosis score charts compared to microbiologically confirmed pulmonary TB case? 1.5 Objectives Broad Objective To evaluate paediatric tuberculosis score charts in the diagnosis of pulmonary TB against culture or Xpert MTB/RIF assay performed on induced sputa from children suspected to have TB in the pediatric departments of BMC Specific Objectives 1. To determine the proportion of patients diagnosed with pulmonary tuberculosis among children suspected to have pulmonary TB in the peadiatric department of BMC. 4

23 2. To determine the sensitivity and specificity of pediatric TB score charts against microbiologically confirmed pulmonary TB cases among children suspected to have TB in the peadiatric department of BMC. 3. To determine the baseline characteristics which are associated with bacteriologically confirmed pulmonary TB among children suspected to have PTB in the peadiatric department of BMC. 4. To assess the utility of sputum induction performed among children suspected to have pulmonary TB in the Peadiatric department of BMC. 5

24 2.0 LITERATURE REVIEW 2.1 Difficulties in Diagnosing Pulmonary TB in children The difficulties in diagnosing peadiatric pulmonary TB (PPTB) have been known for decades. The main contributing factors for diagnostic difficulties are the nonspecificity of signs and symptoms, pauci bacillary nature of PPTB, problem with specimen recovery methods and limitations of the diagnostic test itself (7). Symptoms of TB in children are often non-specific, and a significant proportion of paediatric patients may be asymptomatic during the initial stages of the disease. Among those who are symptomatic the symptoms may include cough, fever, weight loss, night sweats, and wheezing. Several studies have been done to attempt to determine signs and symptoms that can be used for PPTB diagnosis but results showed poor sensitivity and specificity because the symptoms of PPTB can be similar to symptoms of other diseases such as pneumonia, asthma and congenital pulmonary diseases (7,16). 2.2 Pediatric TB Score chart The clinical presentation of pulmonary tuberculosis is often non-specific and there is increased variability in the interpretation of radiological findings. These variabilies become even more evident in the presence of HIV co-infections when other opportunistic infections present with overlapping clinical and radiological findings (6,14,15). To improve the diagnostic accuracy several pediatric Tuberculosis Score Charts (TSC) have been designed for resource limited countries to facilitate the diagnosis of 6

25 TB in children. TSC are mainly checking the combinations of clinical and radiological evidence of disease in the presence of a positive history of TB contact or a positive TST result. Several studies have described the performance of those scoring system (6,7,12 15,17,18); however those systems are poorly validated and cannot be generalized in different epidemiological settings and have not been adapted for use in HIV endemic areas (6,13). The Keith Edwards score chart has been recommended by the Tanzania NTLP for the diagnosis of TB in children (Tanzania National Guideline for Management of TB in Children, 2012) and is commonly used at BMC (BMC treatment guidelines). This score was developed in Papua New Guinea (PNG) and is based almost entirely on clinical findings; microbiological tests and radiology results are not included in the main score, as it is argued that these investigations are not widely available. This score was designed for the diagnosis of both pulmonary and extra pulmonary TB. The original study proposes a score >7 being suggestive of TB based on clinical experience, but this is not validated (12). Studies that have been done to evaluate the performance of Keith Edwards score chart have shown good sensitivity and specificity if applied to children not infected with HIV (17,18); however, in HIV endemic areas it has been shown to have very low specificity (14,19). The Fourie score was designed in 1993 when the International Union Against Tuberculosis and Lung Disease (IUATLD) appointed a task group to develop a scoring system which will show more acceptable sensitivity, specificity, and positive predictive values. The initial assessment of the Fourie score showed a sensitivity and specificity of 41% and 44%, respectively, for young children (0 4 years) compared to older children (5 14 years) with sensitivity and specificity of 62% and 50% 7

26 respectively (20). They therefore proposed the score to be applied as screening tool to select individuals with a high probability of tuberculosis for further examination at a referral centre. The Brazilian MOH score was mainly designed for the diagnosis of pulmonary TB in children and was once evaluated against pulmonary TB cases diagnosed by culture and clinical features together; its sensitivity and specificity were 89% and 86% respectively (21). 2.3 Specimen Recovery Methods Gastric Lavage: Although rarely performed at BMC, three consecutive early morning gastric lavages (GL) is the most common recovery method used to obtain specimen for mycobacteriological confirmation in limited resource countries. In addition to low culture yield, GL has many disadvantages:- specimens must be taken on three sequential days early every morning before breakfast to achieve optimal yields. Furthermore, the procedure itself is unpleasant for the child, semi invasive and usually requires hospitalization as well as additional training of the staff. Therefore, GL is difficult to be done on an outpatient basis where care for most TB suspected children are usually provided. In addition, GL may not be optimal for collection of these specific samples because it is known that a low ph kills tuberculous bacilli (22) and this may affect the growth in subsequent culture media. In addition, peristalsis movement in the evening and while the patient is sleeping makes it likely that many of the TB organisms may have left the stomach by the time of specimen collection. Sputum Induction: Sputum induction has been successfully used in several studies as an alternative to GL to obtain specimen for microbiologic confirmation of PTB in 8

27 children. After pretreatment with an inhaled bronchodilator, nebulization with hypertonic (3% 5%) saline is performed, and secretions are obtained by suctioning or by expectoration in older children (8,11,23). Precautions must be taken to prevent nosocomial transmission during sputum induction. The procedure should be performed in a well ventilated room equipped with UV lighting or in the open air, and sufficient time should be allowed between procedures (6). Appropriate particulate respirators (N95 or FFP2) should be used by staff. Sputum induction has been shown to be feasible, safe, and effective even in infants of one month of age (8,11,23). Two studies involving hospitalized infants in a tertiary care facility in South Africa successfully obtained samples from 95% of the children. In the first study (8), one induced sputum sample yielded more positive culture results (10% of samples) than three sequential GL samples (6% of samples), in the second study (23), the cumulative yield from three induced sputum samples (87%) was greater than that of three GL samples (65%). One induced sputum sample was equivalent to three GL samples (23) and the yield was similar in both HIV infected and HIV uninfected children. Sputum induction has a number of advantages over GL as it can be performed as an outpatient procedure, it is relatively easy to perform, and the yield is higher. The remained challenge is to change the perception of health care workers that microbiologic confirmation of PPTB is not possible and to implement sputum induction as a routine investigative procedure in all health care facilities Alternative Methods of Specimen Recovery: Alternative methods used for specimen recovery include nasopharyngeal aspiration (NPA), bronchoalveolar lavage 9

28 (BAL) and string test to obtain stomach contents. NPA is achieved by passing a canula through one nostril into the nasopharynx; it is minimally invasive and easy to perform. A previous study done in Uganda suggested that the culture yield from NPA was similar to that from sputum induction (24% vs 22%) (9). However, in two studies from Yemen (24) and Peru (25) the culture yields from NPA were low. BAL is a resource intensive and invasive procedure and therefore not recommended for microbiologic confirmation of tuberculosis in resource limited settings. The string test has not been well studied in a pediatric population and does not seem suitable for young children. 2.4 Confirmation of Pulmonary TB in children Confirmation of Mycobacterium tuberculosis (MTB) in pulmonary TB can be performed by (direct or indirect) smear microscopy, by culture or by identification of M. tuberculosis cellular components such as nucleic acid or cell wall component in biological sample. The introduction of fluorescent microscopy with Auramine staining has improved the sensitivity of microscopy and reduced operator time when compared with traditional acid-fast stains, such as Ziehl-Neelsen. However, this advance is unlikely to significantly improve the performance of smear microscopy which currently detects only 10-15% of tuberculosis cases in children (26). Sputum culture on solid media is the current reference standard for the confirmation of pulmonary TB. Despite the high specificity which sputum culture can achieve in the diagnosis of pulmonary TB if combined with a molecular speciation test, it is thought to have relatively poor sensitivity (30% to 40%) when compared to a clinical reference standard in children (26). The other limitations of the traditional culture 10

29 technique are slow turnaround time and the need for highly trained personnel, making it difficult to use as a routine test in a setting with limited resources. The current recommendation by WHO is that Xpert MTB/RIF assay may be used as initial diagnostic test in all children suspected of having PTB and supports the use of a single sputum specimen for diagnostic testing. The sensitivity of this test is better than smear microscopy and is comparable to culture on solid media (27). In addition, it simultaneously detects resistance to rifampicin which is a marker for MDR tuberculosis. However, smear microscopy and culture remain essential for monitoring of therapy for those who are on TB treatment and for monitoring MDR TB as it was shown for adults, that Xpert MTB/RIF assay remains positive for a long time even in successfully treated TB cases (28). 11

30 3.0 MATERIALS AND METHODS 3.1 Site Description This study was conducted in the department of paediatric and child health at Bugando Medical Centre, Tanzania. BMC is a consultant and teaching hospital for the Lake and Western zones of the United Republic of Tanzania. Situated along the shores of Lake Victoria in Mwanza, it has 900 beds and it serves both inpatient and outpatients. It is a referral centre for tertiary specialist care for eight regions, including Mwanza, Geita, Simiu, Mara, Kagera, Shinyanga, Tabora and Kigoma. It serves a catchment population of approximately 13 million people. The department of peadiatrics and child health has a capacity of 121 beds. The department is subdivided into general peadiatric wards, malnutrition wards, semi intensive care unit, neonatal unit, neonatal intensive care unit and outpatient department. 3.2 Study design A cross sectional hospital based study was conducted over a five-month period from October 2013 to April Sample size Sample size was estimated using the Buderer s formula (29), to determine the sensitivity(s N ) and specificity(s P ) of 90% with absolute precision of less than 10% at a 95% confidence interval [CI] with a 18.9% prevalence of PTB among TB suspects in children(11). 12

31 Sample size (n) based on specificity= Z 2 1 α/2 SN (1 SN) L 2 Prevalence Sample size (n) based on specificity = Z 2 1 α/2 SP (1 SP) L 2 (1 Prevalence) where n = required sample size, S N = anticipated sensitivity, S P = anticipated specificity, α = size of the critical region (1 α is the confidence level), z 1-α/2 = standard normal deviate corresponding to the specified size of the critical region (α) L = absolute precision desired on either side (half-width of the confidence interval) of sensitivity or specificity. Based on the above formula the minimum sample size (n) was estimated to be 186 children suspected to have PTB. 3.4 Study Population Children aged 2months to 12 years with respiratory TB symptoms as defined by Graham et al (1) who were seeking care in the paediatric department of BMC, and met the inclusion criteria were enrolled into the study. Inclusion criteria Written informed consent Any one of the following: Persistent (> 2 weeks), non-remitting cough 13

32 Persistent (> 1 weeks) and unexplained fever (38 0 C) Unexplained weight loss/failure to thrive: 5% reduction in weight compared with the highest weight recorded in last 3 months History of TB contact within the preceding 24 months Exclusion criteria Patient on TB treatment Patient on Isoniazid Prophylaxis Treatment (IPT) Known asthmatic or expiratory wheezes on auscultation 3.5 Data collection Standard diagnostic procedures The enrolment of the children suspected to have pulmonary TB included the delivery of study information and obtaining an informed consent, the medical history of participants, a physical examination with collection of detailed anthropometric data such as middle upper arm circumference for children above 6 months (MUAC), weight, and height. Furthermore HIV testing and chest radiography was part of the enrolment visit. All children aged 2 months to 12 years admitted to the peadiatric at wards were screened for TB symptoms. Parents or guardians of those who fulfilled the criteria were asked to consent for enrollment into the study. Children suspected to have PTB who were referred from the outpatient clinic were also enrolled once they fulfilled the eligibility criteria. 14

33 All enrolled children were re-assessed using the Keith Edward, MASA (30), IUATLD or Fourie (20) and Brazilian MoH (31) diagnostic charts. Details of these TB score charts are provided in appendix 5. Chest X-Ray was taken in frontal view and was read by an independent radiologist who was blinded to the study. A lateral view CXR was taken only when suggested by the radiologist. CXR findings were reported in a standardized form with predetermined terminology to describe CXR abnormalities (32) (Appendix 2). Chest X-rays were defined as consistent with tuberculosis if there was a positive response for any one of the radiological features. A TST, using intradermal injection of 5 tuberculin units of purified protein derivative (Mantoux) (TUBERSOL, Sanofi Pasteur Limited, Canada), was performed by trained personnel on the volar aspect of the left forearm. The transverse diameter of induration was measured in millimetre after 48 to 72 hours. A positive TST was regarded as a measurement of 10mm in HIV-uninfected children, and 5mm in HIV-infected children (actual measurement in mm was recorded) (Appendix 6). One sputum specimen was obtained by sputum induction (Appendix 4) which was done by the investigator. A Pulmo Mist II nebulizer (Nidek Medical, USA) delivered 5 ml of 5% sterile saline solution for 10 to 15 minutes. Salbutamol solution 2.5mg was added to the nebulizer solution to prevent bronchoconstriction. Sputum was obtained by suctioning through the nasopharynx with a sterile mucus extractor of catheter size 7 or 8. Specimens were transported directly to the laboratory for processing. New sterile disposable nebulizer masks, medication tanks and tubing were used for each patient. Non disposable parts (tube connecting suction machine to mucus trap) were cleaned and decontaminated after every use by submerging in 2% 15

34 glutaraldehyde for 20 minutes and allowed to air-dry. Data from a previous study shows that parts of sputum induction equipment which are highly contaminated with M. tuberculosis can effectively be decontaminated with glutaraldehyde (33). Information about the HIV status was extracted from hospital patient files. Those with no results were asked to have an HIV test, by using an HIV rapid test according to the algorithm of the National HIV/AIDS guideline (Appendix 6.8). Information about other diseases and tests not included in this study were extracted from BMC patient files. All admitted children were followed up for resolutions or persistence of TB symptoms until discharge from hospital and then two months after being discharged through phone calls. Children from the outpatient clinic were tellephonically followed up for any treatment they were receiving for up to two months. During follow up, TB symptoms (persistent cough, persistent fever, weight loss, etc) were specifically asked for and those who were still experiencing symptoms after two months were advised to come back to the clinic for re-assessment. No follow up was done after two months and patients who were still experiencing symptoms were advised to continue follow up and further workup through BMC outpatient clinic Laboratory procedures All laboratory procedures were performed at the routine medical microbiology laboratory of the Bugando Medical Centre. Sputum specimens were processed for Fluorescent smear microscopy with Auramine stain to detect AFB in sputum. Smear microscopy were read as follows; 1-9 AFB per 100 scanned fields were recorded in absolute number, AFB per 100 scanned 16

35 fields were reported as (1+), 1-10 AFB per field scanned were reported as (2+) and >10 AFB per field scanned as (3+). TB PCR (Xpert MTB/RIF assay) was also performed on sputum samples when GeneXpert MTB/RIF cartridges were available. After microscopy and Xpert MTB/RIF assay, the remained sputum samples were processed for LJ culture. At the beginning of this study we did not have LJ culture media available, therefore all the samples collected from October to December (30 patients) and sputum samples collected from January to December (40 patients) were stored at C for up to four weeks while waiting for LJ culture media to arrive. Sputa were then decontaminated by modified Petroff s methods using 4% sodium hydroxide (NaOH) and then concentrated by centrifugation at 3000g. After centrifugation it was cultured onto slopes of Lowenstein Jensen (LJ) medium with glycerol (GLJ) and pyruvate (PLJ), and incubated at 37 o C for 8 weeks. Each sputum sample was inoculated in 2 LJ slopes. Culture slopes were inspected on weekly a basis to observe growth Study duration and management of study subjects Recruitment was done over a period of 5 months from October 2013 to April All treatment decisions were taken independently of the study classification by the clinicians who were taking care of the patient. However, laboratory study results were made available to the attending clinicians in order to assist the treatment decision. In the case of positive cultures, patients were called back after culture results to be informed of their results and referred to the TB clinic for treatment, if TB treatment had not yet been initiated. 17

36 3.6 Outcome of the study The primary outcome of this study was defined as the number of microbiologically confirmed TB cases. Secondary outcome was defined as the number of total TB cases identified according to the diagnostic classification and the number of sputum inductions performed. 3.7 Study Variables Independent Variables Age Sex TB contact HIV status Dependent Variables Positive sputum culture Positive sputum smear microscopy Positive Xpert MTB/RIF Positive TB score Duration of TB symptoms Weight for age Weight for height/length TST results BCG vaccination/scar Chest X-ray findings 3.8 Statistical analysis Completed interview schedule was coded by numbers and entered into computer software MS Excel Cross-checking and data cleaning was done. The data were then transferred to STATA version 13 (Stata Corp, Inc., College Station, TX, USA) software for analysis. Variables were summarized as proportions for categorical variables and median with IQR for continuous variables. Associations between 18

37 variables were explored by logistic regression or Fischer s exact test where appropriate. All statistical tests were considered significant if the two sided P-value (p) was <0.05. Sensitivity, specificity, positive predictive (PPV) value, and negative predictive value (NPV) of Keith Edward score, MASA score, Fourie Score and Brazilizn MOH score were calculated against pulmonary TB case by using 2 by 2 contingency table. 3.9 Data quality and assurance The investigator interviewed and examined all patients. All information obtained from the patients were recorded in structured questionnaires and kept in a hard cover file. The files were stored by the investigator. Only the investigator and study team had access to patient information. Questionnaires were then coded by numbers and entered in computer software MS Excel Cross-checking and data cleaning were done. During data cleaning and cross checking missing information were obtained by going back to the structured questionnaires and lab results. The data were then transferred to STATA version 13 (Stata Corp, Inc., College Station, TX, USA) software for analysis Ethical consideration Ethical clearance and approval were obtained from the joint CUHAS and BMC Ethics review board. Written informed consent was obtained from parents/guardians of the study participants. Witnessed oral consent was obtained from illiterate parents or guardians in the presence of impartial witness. Confidentiality was assured and unauthorized persons had no access to the collected data. Each study subjects was 19

38 assigned study identification number, which were kept confidential throughout the study period. 20

39 4.0 RESULTS 4.1 Baseline Characteristics From October 2013 to April 2014 a total of 1032 patients were screened for enrollment in the study. Almost all screenings (98.4%) were inpatients admitted to the general peadiatrics and malnutrition wards of BMC. A total of 197 (19.1%) children were eligible for enrollment in the study. A total of 192 (97.5%) patients were enrolled in the study; 5 (2.5% of all eligible patients) patients were excluded due to unstable medical conditions which made it impossible to investigate the child and perform induced sputum Of the 192 children enrolled in the study. One hundred and three (53.7%) were male. Median age among participants was 1.9 years (IQR ). Only 4 (2.1%) participants had a history of known TB contacts. Almost all children (92.7%) had received BCG vaccination at birth. About half of the patients (49.5%) had severe acute malnutrition. HIV test were positive in 15.1% of all participants while 9.4% were HIV exposed children (Table 1). 21

40 Table 1: Baseline characteristics of study participants (n=192) Characteristics Number (%), Median [IQR] Sex Male 103 (53.7) Age in years (Median) 1.9 [ ] Below 2years 100 (52.1) 2 years - <5 years 47 (24.5) 5 years and above 45 (23.4) TB contact Yes 4 (2.1) No 176 (91.7) Unknown 12 (6.3) BCG vaccine Yes 178 (92.7) No 13 (6.8) Unknown 1 (0.5) Nutrition Status Normal 48 (25.0) Mild acute malnutrition 25 (13.0) Moderate acute malnutrition 24 (12.5) Severe acute malnutrition 95 (49.5) Weight for age Normal 38 (19.8) Mild underweight 33 (17.2) Moderate underweight 28 (14.6) Severe underweight 93 (48.4) TST Positive 22 (11.5) HIV status HIV positive 29 (15.1) HIV exposed** 18 (9.4) HIV negative 129 (67.2) Unknown HIV status 16 (8.3) Type of patients Inpatient 175 (91.2) Outpatient 17 (8.8) **Children below 18 months of age who had positive HIV antibody test but not yet confirmed by DNA PCR 22

41 Figure 1: Study flow diagram 1032 patients were screened for enrollment into the study from October 2013 to March (98.4%) inpatients 17 (1.6%) outpatients 807 (78.2%) did not meet eligibility criteria 28 (2.7%) died before being screened 197 (19.1%) we eligible for enrollment 192 (97.5%) enrolled in the study 5 (2.5%) excluded because of critical medical condition 175 (91.1%) inpatients 17 (8.9%) outpatients 5 (2.6%) not tested due to lack of sputum sample 187 (97.4%) tested by fluorescence microscopy and by LJ culture 73 (39.1%) not tested due to shortage of cartridges 114 (60.9%) tested by Xpert MTB/RIF assay 23

42 4.2 Specimen recovery Sputum specimen was obtained in 187 (97.4%) patients. A single sputum induction was successfully performed in (183) 95.8% patients, in 3 (1.6%) patients sputum specimen was obtained by nasopharyngeal aspiration because they were not able to tolerate the sputum induction procedure. Sputum specimens were not obtained in 5 (2.6%) patients. The average volume of sputum obtained was 2.4 mls (range ). Epistaxis was the only adverse event reported among participants and was experienced in 25 (13.4%) patients. Epistaxis stopped soon after the procedure without any intervention in all cases. No serious complications occurred after the procedure. 4.3 Common presenting symptoms Cough, fever, and weight loss were the most common TB symptoms reported among the participants with 96.6% (185 patients), 94.8% (182 patients) and weight loss 69.8% (134 patients) respectively. Hemoptysis constituting 0.3% (5 patients) was the least symptoms reported (Figure 2). 24

43 Figure 2: Common presenting symptoms among patients enrolled in the study 100 Percent (Proportion) of patients Cough Fever Weight loss Fatigue Night sweat Difficulty in breathing Chest pain Hemoptysis 4.4 Radiological Characteristics Chest X-ray in frontal view was obtained in all participants. One hundred and thirteen (58.8%) had abnormal chest X-ray findings, of them 53 (46.9%) had findings consistent with TB. Airspace opacification and hilar lymphadenopathy were the most common radiological findings presenting in 27 (50.0%) patients and 20 (37.0%) patients respectively in patients with features consistent with pulmonary TB. Airway compression (1 patient) and vertebral spondylitis (1 patient) each constituting 1.8% were the least reported radiological findings. Other radiological findings were miliary pattern, pleural effusion and cavities which were present in 12 (22.2%), 11 (20.4%) and 11 (20.4%) respectively. 25

44 4.5 Patients Follow-up At enrollment 38 (19.8%) were eligible for initiation of TB treatment; 36 (94.7%) of those who were eligible started on ant TB medication, 2 (5.3%) died before treatment initiation, 1 (2.6%) had anti TB stopped after having alternative diagnosis confirmed. At 2 months follow up 4 (2.1%) patients who were not on TB medication were found eligible for TB treatment; 2 of them were started on TB treatment, 1 died and 1 was lost to follow up. There were 18 (9.4%) patients who died after enrollment; among those who died 7 (38.9%) patients were on TB treatment. At 2 months follow up there were 20 (10.4%) deaths. All deaths occurred in patients who were admitted. Twenty three patients (12.0%) were lost to follow up (Table 2). Table 2: Follow up among patients enrolled in the study PTB n (%) No PTB n (%) Total (%) Two months follow up for previously admitted patients No TB symptoms 22 (62.9) 100 (71.4) 122 (69.7) TB symptoms 2 (5.7) 11 (8.4) 13 (7.4) Died 10 (28.6) 10 (7.14) 20 (11.4) Loss to f/u 1 (2.9) 19 (13.4) 20 (11.4) Sub total 35 (100.0) 140 (100.0) 175 (100.00) Two months follow up for patients referred from outpatients clinics No TB symptoms 4 (80.0) 8 (66.7) 12 (70.6) TB symptoms 1 (20.0) 1 (8.3) 2 (11.8) Loss to f/u 0 (0.0) 3 (25.0) 3 (17.6) Sub total 5 (100) 12 (100) 17 (100) 26

45 4.6 Yield for Mycobaterium tuberculosis from one induced sputum Sputum was obtained from 187 (97.4%) patients. There were 10 (5.3%) patients whom PTB were confirmed by culture and/or Xpert MTB/RIF assa. Yield for MTB was 1.1%, 4.3% and 2.6% from sputum smear microscopy, sputum culture and Xpert MTB/RIF assay respectively (Table 3). Among patients who had bacteriological confirmation of pulmonary TB, 1 patient had confirmation by both positive culture and Xpert MTB/RIF assay, 2 patients had confirmation by positive Xpert MTB/RIF assay while culture was negative, and in the remaining 7 patients confirmation was by positive culture alone. Half (4 patients) of those with positive culture had no GeneXpert performed. Fluorescent AFB smear was positive in 2 patients among them one had both positive culture and positive Xpert MTB/RIF assay while the other had positive culture and GeneXpert not performed. Table 3: Yield for MTB from one induced sputum among patients enrolled in the study Total Positive cases n (%) Sputum smear (1.1) Culture (4.3) Xpert MTB/RIF (2.6) 4.7 The proportion of PTB cases The overall proportion of pulmonary TB cases was 20.8% (40 patients) among patients who were enrolled in the study. The proportion of confirmed Pulmonary TB was 5.2% (10 patients). There were 10 (5.2%) with probable PTB and 20 (10.4%) patients with possible PTB. 27

46 About 64% of all the patients had a score suggestive of likey TB based on Fourie score, 22.9% of the patients had likely TB based on Keith Edward score while very few (3.6%) had a score suggestive of suspected TB by MASA score (Table 4). Table 4: TB cases based by Peadiatrics TB score charts and Diagnostic classification among patients enrolled in the study (N = 192) n (%) Keith Edward Score MASA score Fourie TB Likely 44 (22.9) TB Unlikely 148 (77.1) Suspected TB 7 (3.6) Not suspected TB 185 (96.4) TB likely 122 (63.5) TB unlikely (36.5) Brazilian MOH score Very likely PTB 14 (7.3) Possible PTB 42 (21.9) Unlikely PTB 136 (70.8) Diagnostic Classification Confirmed PTB 10 (5.2) Probable PTB 10 (5.2) Possible PTB 20 (10.4) Unlikely PTB 37 (19.3) No TB 115 (59.9) 4.8 Sensitivity, Specificity, & Positive/Negative predictive values All scores showed low sensitivity against microbiologically confirmed PTB case (10% - 50%). MASA scores showed higher specificity of 97.2% but lowest sensitivity, Fourie score showed very low specificity of 34.5%. Positive predictive 28

47 values ( %) were very low for all scores; negative predictive values (NPV) ranged from 92.4% to 96.3% for all scores (Table 5). Table 5: Sensitivity, specificity, PPV and NPV of TSCs against microbiologically confirmed pulmonary TB (n=10) Positive TB Confirmed Score Charts TB case Sv (%) Sp (%) PPV (%) NPV (%) Keith Edward MASA Fourie Brazilian MOH Sv = Sensitivity; Sp = Specificity; PPV = Positive predictive value; NPV = Negative predictive values. The calculations were based on a total 187 patients whom sputum samples were obtained. 4.9 Factors associated with microbiologically confirmed PTB There were no baseline characteristics which had significant association with confirmed pulmonary TB (Table 6). Airspace opacification, cavities and vertebral spondlytis on chest X-ray were significantly associated with confirmed pulmonary TB with p-values of <0.01, 0.01 and 0.05 respectively (Table 7). In addition, chest X-ray with any finding consistent with pulmonary TB was significantly associated with confirmed pulmonary TB case. 29

48 Table 6: Baseline characteristics associated with confirmed TB among patients enrolled in the study Characteristics Confirmed TB (%) No TB (%) P-value Sex Male 4 (40.0) 97 (54.8) 0.52 Female 6 (60.0) 80 (45.2) Age (years) < 5 years 8 (80.0) 40 (22.6) 5 years 2 (20.0) 137 (77.4) 1.00 TB contact Yes 1 (10.0) 3 (1.7) No 9 (90.0) 165 (98.3) 0.20 BCG vaccine Yes 9 (90.0) 165 (93.2) No 1 (10.0) 11 (6.2) 0.52 Unknown 0 (0.0) 1 (0.6) Nutrition status Severe malnutrition 5 (50.0) 88 (49.7) No severe malnutrition 5 (50.0) 89 (50.3) 1.00* Weight for age Severe underweight 4 (40.0) 86 (48.6) 0.75 No severe underweight 6 (60.0) 91 (51.4) TST Positive 3 (30.0) 18 (10.2) Negative 7 (70.0) 159 (89.8) 0.09 HIV status Positive 1 (10.0) 27 (27.2) Negative 7 (70.0) 118 (66.7) HIV exposed 1 (10.0) 17 (9.6) 1.0 Unknown status 1 (10.0) 15 (8.5) *p-value was calculated by chi-square test. In all other variables p-values were calculated by Fischer s exact test. 30