PROTOCOL FOR SUBMISSION OF THESIS FOR THE DEGREE OF MD (PEADIATRICS) UNIVERSITY OF DELHI

(SESSION 2012-2015)
Title Of Thesis : EVALUATION OF ROLE OF INDUCED SPUTUM IN DIAGNOSIS OF CHILDHOOD PULMONARY TUBERCULOSIS. : Lady Hardinge Medical College And Associated Hospitals, New Delhi-110001 : : Dr. VIKAS KUMAR GAUTAM : : Dr. VARINDER SINGH Professor , Department of Pediatrics Lady Hardinge Medical College & Associated Hospitals, New Delhi : : Dr. KAMAL CHOPRA / Dr. HANIF New Delhi Tuberculosis Centre New Delhi :

Name of the Institution Signature Name of the Student Signature Name of the supervisor

Signature Name of the Co-Investigator

Signature

Name of Head of the Department : Dr. S. ANEJA Director and Professor Department of Pediatrics Lady Hardinge Medical College & Associated Hospitals, New Delhi Signature :

Name of Head of the Institution : Dir. Prof. ATUL MURARI Principal and Medical Superintendent Lady Hardinge Medical College & Associated hospitals, New Delhi

LADY HARDINGE MEDICAL COLLEGE & SMT.S.K.HOSPITAL NEW DELHI

-UNDERTAKINGI/we agree to abide by ethical guidelines for biomedical research on human subject (As Per ICMR guidelines) while conducting the research project being submitted for ethical committee consideration. Project is considered to be absolutely essential for the advancement of knowledge and for the benefit of all. Only subjects who volunteer for the project will be included. Their informed consent shall be obtained prior to commencement of the research project and subjects will be kept fully appraised of all consequences. Privacy and confidentiality of the subjects shall be maintained and without the consent of the subject, no disclosure will be made. Proper precautions shall be taken so as to minimize risk and prevent irreversible adverse effects. Professionally competent persons will conduct research. Research will be conducted in a fair, honest impartial and transparent manner. Research will be accountable for maintaining proper records. Research will be conducted in keeping in view the public interest at large. Research reports, materials and data will be preserved (as per institutional guidelines). Result of research will be made known through scientific publications. Professional and moral responsibilities will be of the researchers, directly or indirectly connected with the research. Only those drugs, which are approved by the Drug controller of India for a specific purpose, will be used in the research project. Signature: Supervisor Dr. VARINDER SINGH Professor Department of Pediatrics LHMC and Associated Hospitals, New Delhi

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Signature: Name of the Investigator Dr. VIKAS KUMAR GAUTAM Department of Pediatrics LHMC and Associated Hospitals New Delhi.

CERTIFICATE FROM INSTITUTION

I, Dr. Vikas Kumar Gautam, hereby declare that to the best of my knowledge, no study has been carried out on the topic- Evaluation of Role of Induced Sputum in diagnosis of Childhood Pulmonary Tuberculosis over the past five years in Delhi University.
Signature: Name of Candidate: Dr. Vikas Kumar Gautam Department of Pediatrics Lady Hardinge Medical College and Associated Hospitals New Delhi-110001 Signature: Name of supervisor: Dr. Varinder Singh Professor Department of Pediatrics Lady Hardinge Medical College and Associated Hospitals New Delhi-110001

INTRODUCTION
Tuberculosis is one of the most significant health problems in developing countries which causes more preventable deaths than any other infectious disease. It is one of the top ten causes of death among children worldwide. Diagnosis of pulmonary tuberculosis is difficult in young children in whom clinical and radiological signs can be non-specific. Microbiological confirmation of tuberculosis is desirable for definitive diagnosis. Such a proof is often lacking in childhood tuberculosis because of difficulty in collection of sputum due to less sputum production, less expectoration and closed lesions. In young children, culture confirmation has relied on specimens from sequential gastric lavages. Limitations of gastric lavage, however, include the need for an overnight fast, repeated specimens, and admission of children. Moreover, the procedure is time consuming and unpleasant for both child and health worker. Although, sputum induction has been successfully used in adults but this technique has been recently investigated in young children largely in South Africa. This technique is only not regarded as feasible in young children since they swallow their sputum and do not expectorate. However, adding the nasophrangeal suction after induction has made the procedure more suitable for children. Sputum induction is also considered less invasive than lavage, can be done even in resource-poor settings and also as outpatients procedure. While there are few studies from S Africa supporting the use of IS for diagnosis, the same results have not been reported from a study in our country. The availability of a specimen by a feasible easier method is a welcome step but by itself, this may not be adequate as the smear positivity rate for pediatric pulmonary disease are about 10-12% only. The use of direct (unconcentrated) sputum smear microscopy is the primary method of tuberculosis diagnosis in the developing world. Traditional Ziehl-Neelsen (ZN) Staining uses a light or bright field microscopy at 100X magnification. Light-Emitting Diode (LED)-fluorescence microscopy in which auramine based staining causes the acid-fast bacilli to fluoresce against a dark background is widely used in many parts of the world and was compared with Ziehl-Neelsen (ZN) staining using culture as the gold standard suggest that LED-FM is more sensitive than conventional light microscopy, but specificity was comparable to ZN microscopy. Additionally, yield of positivity with this technique in pediatrics population is not known. If the addition of LED-FM increases the smear positivity yield for an easily collected specimen, it has the potential to aid quality diagnosis of TB among children. The Xpert MTB/Rif test is a rapid cartridge-based fully automated NAAT (nucleic acid amplification test) for TB case detection and rifampicin resistance testing, suitable for use in disease-endemic countries. It purifies, concentrates, amplifies (by rapid, real-time PCR) and identifies targeted nucleic acid sequences

in the TB genome, and provides results from unprocessed sputum samples in less than 2 hours, with minimal hands-on technical time. Although molecular amplification is already a proven technology in diagnosis, other existing testmethods are too complex for routine and widespread use in developing countries. The need for sample processing and DNA extraction adds another level of complexity to implementation in settings where resources are limited. GeneXpert, the test device platform, was launched by Cepheid in 2004 and simplifies molecular testing by fully integrating and automating the three processes (sample preparation, amplification and detection) required for real-time PCR-based molecular testing. The Xpert MTB/RIF test is currently the only molecular test of its kind and uses a cartridge containing all elements necessary for the reaction, including lyophilized reagents, liquid buffers and wash solutions. Target detection and characterization is performed in real time using a six-colour laser detection device. This is a relatively new test where the reported sensitivity is far greater than the smear examination. It may be worthwhile to know if this improved sensitivity- largely investigated among adult cases- is also seen as well with childhood cases. Equally relevant is to know whether the alternate easily accessible specimens like IS or stool can be used for a NAAT based test.

LACUNAE IN KNOWLEDGE

Few sStudies comparing gastric lavage with sputum induction in children adults with suspected tuberculosis have reported that the diagnostic yield from sputum is higher. However, most of these are from S Africa. As the disease profile in S Africa is different due to a significant proportion of HIV co-infected patients, it is important to evaluate the feasibility and yield of this method in our settings. few studies of sputum induction in young children are available. The Gastric Lavage (GL) method is preferred in diagnosis of Tuberculosis in children who swallow their sputum and cannot expectorate. However, this method has reportedly yielded varied results in different studies. Moreover Gastric Lavage needs overnight stay while induced sputum can be done on OPD basis. Most of the Data is from South Africa where Tuberculosis/HIV co-exist but it is unclear whether the difference in disease profile may affect the yield. Further, aAlthough LED-FM has significant benefits over ZN Microscopy, the additional yield for paucibacillary pediatric sample has not been studied. Cartridge based NAAT has a potential to replace smear examination especially among children due to its reported better yield in smear negative cases. However, there is little data for its use in children, particularly using GA or IS or stool as a sample.

REVIEW OF LITERATURE
For diagnosis of pulmonary tuberculosis in childhood patients, sequential gastric lavages are recommended. Sputum induction has been successfully used in adults but this technique has been recently investigated in young children largely in South Africa.. In a study conducted by Prof. H J Zar et al on 250 children aged between 1 month-5 years with suspected TB, , sputum induction and gastric lavage were done on three consecutive days following a standard procedure. Specimens were stained for acid-fast bacilli and each sample was cultured singly for M tuberculosis. A positive smear or culture for M tuberculosis was obtained from 62 (25%) children; of these, 58 (94%) were positive by culture, whereas almost half (29 [47%]) were smear positive. Samples from induced sputum and gastric lavage were positive in 54 (87%) and 40 (65%) children, respectively (difference in yield 5.6% [1.4-9.8%]. Tthe yield from one sample from induced sputum was similar to that from three gastric lavages.. All sputum induction procedures were well tolerated; minor side-effects were increased coughing, epistaxis, vomiting, or wheezing. The authors concluded that Induced sputum is preferable to gastric lavage for diagnosis of pulmonary tuberculosis in infants and children. There were 250 children aged between 1 month-5 years included in the study. Sputum induction and gastric lavage were done on these children on three consecutive days following a standard procedure. Specimens were stained for acid-fast bacilli and each sample was cultured singly for M tuberculosis. A positive smear or culture for M tuberculosis was obtained from 62 (25%) children; of these, 58 (94%) were positive by culture, whereas almost half (29 [47%]) were smear positive. Samples from induced sputum and gastric lavage were positive in 54 (87%) and 40 (65%) children, respectively (difference in yield 5.6% [1.49.8%].(Zar HJ, Hanslo D, Apolles P, Swingler G, Hussey G. Lancet. 2005 Jun 4-10;365(9475):1926.) HOW MANY WERE HIV+???

In aAnother study by the same group, was conducted by Zar HJ, on the same subject matter on 149 children concluded that Sputum induction can be safely and effectively performed in infants and young children. Induced sputum provides a satisfactory and more convenient specimen for bacteriological confirmation of pulmonary tuberculosis. Sputum induction was successfully performed in 142 children out of 149 children. M tuberculosis cultured in 16 children, grew from induced sputum in 15. Gastric Lavage performed in 142 children, was positive in nine; in eight of these M tuberculosis also grew from induced sputum. The difference between yields from induced sputum compared to Gastric Lavage was 4.3%., (Dr H Zar, Child Health Unit, Arch Dis Child 2000;82:305-308 doi:10.1136/adc.82.4.305) THIS SHOULD PRECEDE AS THIS IS DONE EARLIER In a study conducted at St. Marys Hospital, Uganda, proportion of positive induced sputum cultures among probable childhood tuberculosis cases was determined. 750 children with suspected Tuberculosis were evaluated clinically, by chest radiography and by tuberculin test (Mantoux). Probable cases underwent sputum induction. Induced sputum yielded 12/101 (12%) positive smears of Mycobacterium tuberculosis and 30/101 (30%) positive cultures. Induced sputum yielded 12/101 (12%) positive smears of Mycobacterium tuberculosis and 30/101 (30%) positive cultures. (Iriso R, Mudido PM, Karamagi C, Whalen C. Department of Paediatrics, St Mary's Hospital Lacor, Gulu, Kampala, Uganda, Int J Tuberc Lung Dis. 2005 July St. Marys Hospital, Uganda) A prospective study in South Africa in which a total of 270 children were enrolled with median age 38 months. Sputum induction was successful in 269 (99%) children. 65 (24%) children were clinically diagnosed, of whom 11 (16.9%) were microbiologically confirmed. Sputum induction procedures were well tolerated and no major adverse events were occurred. Sputum induction was useful for making a microbiological diagnosis, increasing the

number of children diagnosed and treated for PTB.

(Moore HA, Apolles P, de Villiers PJ, Zar HJ in South Africa Int.

J Tuberc Lung Dis. 2011 Sep; 15(9):1185-90.) All the above mentioned studies conclude that induced sputum is very much effective and sensitive in diagnosing childhood pulmonary tuberculosis. Induced sputum has less side-effects and also it does not require overnight fasting and can be done on OPD basis. Our study also aims to assess the role of induced sputum in childhood pulmonary tuberculosis.

Fluorescence versus conventional sputum smear microscopy for tuberculosis: Most of the worlds tuberculosis cases occur in low-income and middle-income countries, where sputum microscopy with a conventional light microscope is the primary method for diagnosing pulmonary tuberculosis. A major shortcoming of conventional microscopy is its relatively low sensitivity compared with culture. A systematic review of 45 studies comparing fluorescence microscopy with conventional microscopy suggests that: fluorescence microscopy is more sensitive for the diagnosis of pulmonary tuberculosis than conventional microscopy; and the specificity of fluorescence microscopy for detection acid-fast organisms in sputum is similar to that of conventional microscopy; (Karen R Steingart, Megan Henry, Vivienne Ng, Philip C Hopewell, Andrew Ramsay, Jane Cunningham, Richard Urbanczik, Mark Perkins, Mohamed Abdel Aziz, Madhukar Pa Lancet Infect Dis 2006; 6:57081i) LED-FM had higher sensitivity, lower specificity than ZN smear microscopy for diagnosis of pulmonary TB. which resulted in a significantly higher number of patients with PTB being treated, but lower overall diagnostic accuracy, Recently, however, Cattamanchi et al. suggested that LED-FM had lower specificity than conventional ZN smear microscopy in patients co-infected with HIV in Uganda [17], suggesting that the loss of specificity was more evident in patients who had scanty AFB in sputum. (Cuevas LE, Al-Sonboli N, Lawson L, Yassin MA. et al; Liverpool School of Tropical Medicine, Liverpool, United Kingdom PLoS Med. 2011 Jul;8(7):e1001057. Epub 2011 Jul 12.) LED-FM had a higher sensitivity than ZN smear microscopy, it had a higher rate of false positives. The accuracy of LED-FM after three sputum samples examined was 85%, compared to 91.8% in samples tested using ZN smear microscopy. The researchers suggest that despite this shortcoming, the combined increased sensitivity and shorter examination times might be worthwhile to diagnose more people from TB, (doi:10.1038/nmiddleeast.2011.93; Published online 21 July 2011 Mohammed Yahia)

These Studies show varied results and sensitivity and specificity of LED-FM over ZN smear was not different. There may be some limitations in collection of sputum and the method of processing. The GeneXpert assay was found to be highly specific (99.2%) while the sensitivity of the assay for detecting M. tuberculosis (MTB) DNA was 98,2% in smear-positive patients and 72,5% in smear-negative culture-positive patients. Sensitivity on smear-negative culture-positive patients increased by 12.6% by performing a second GX investigation on the same patient (different sputum). (National Health Laboratory Service 2012)

RESEARCH HYPOTHESIS

Induced Sputum is as sensitive and specific as gastric lavage in diagnosing pulmonary tuberculosis in childhood patients

AIMS AND OBJECTIVES

Primary Objective: To study the role of Induced Sputum in bacillary diagnosis of childhood pulmonary tuberculosis.

Secondary Objective: Role of Light-Emitting Diode Fluorescence Microscopy in childhood pulmonary tuberculosis.

PATIENTS AND METHODS

STUDY DESIGN: Hospital based observational study

PERIOD OF STUDY : November 2012-March 2014

PLACE OF STUDY : Department of Pediatrics, Lady Hardinge Medical College and Associated Kalawati Saran Children Hospital, New Delhi

STUDY POPULATION : Children up to 15 years of age in Kalawati Saran Children Hospital, New Delhi

SAMPLING AND SAMPLE SIZE CALCULATION :

A Convenient sample size of eighty probable pediatrics Tuberculosis cases will be taken

MATERIALS AND METHODS

PATIENTS
INCLUSION CRITERIA All children up to fifteen years of age who have high probability to have Pulmonary Tuberculosis on clinical and radiological basis with/without positive Mantouxs test

EXCLUSION CRITERIA 1. Children who can produce sputum 2. Children earlier diagnosed with tuberculosis 3. Children with wheezing 4. Children showing signs of upper airway obstruction 5. Oxygen saturation less than 92%

METHODOLOGY
For enrollment of children, a written informed consent from parents or legal guardian is required. History and Physical examination of the children will be done.

Two Gastric Lavage samples and two Induced Sputum need to be done on two consecutive days. For taking these samples, the child needs to be admitted in the hospital and put on a fast for two consecutive overnights.

For each sample, Ziehl-Neelsen (ZN) Staining & Light-Emitting Diode (LED) Fluorescence Microscopy.

Singly first sample will be cultured (MGIT* Culture) *Myco Bacteria Growth Indicator Tube

STATISTICAL METHODS

Results of the study will be tabulated and analyzed using standard statistical methods. The sensitivity, specificity and positive and negative predictive values of Induced Sputum and Gastric Lavage smears and cultures calculated.

REFERENCES

(1) Prof Heather J Zar PhD David Hanslo FRCPath a, Patricia Apolles RN a, Prof George Swingler PhD a, Prof Gregory HusseyMMed Induced sputum versus gastric lavage for microbiological confirmation of pulmonary tuberculosis in infants and young children (2) Consensus Statement on Childhood Tuberculosis, Working Group on Tuberculosis, Indian Academy of Pediatrics (3) doi:10.1038/nmiddleeast.2011.93; Published online 21 July 2011 Mohammed Yahia Zar HJ, Hanslo D, Apolles P, Swingler G, Hussey G Lancet. 2005 Jun 4-10;365(9475):1926. (4) H J Zar, Child Health Unit, Arch Dis Child2000;82:305-308 doi:10.1136/adc.82.4.305. (5) Iriso R, Mudido PM, Karamagi C, Whalen C. Department of Paediatrics, St Mary's Hospital Lacor, Gulu, Kampala, Uganda, Int J Tuberc Lung Dis. 2005 Jul;9(7):716-26. (6) Moore HA, Apolles P, de Villiers PJ, Zar HJ.,Int J Tuberc Lung Dis. 2011 Sep;15(9):1185-90. (7) Karen R Steingart Megan Henry Vivienne Ng, Philip C Hopewell, Andrew Ramsay, Jane Cunningham, Richard Urbanczik, Mark Perkins, Mohamed Abdel Aziz, Madhukar Pai et al Lancet Infect Dis 2c006; 6:570-81 Fluorescence versus conventional sputum spear microscopy for tuberculosis (8) Cuevas LE, Al-Sonboli N, Lawson L, Yassin MA. et al; Liverpool School of Tropical Medicine, Liverpool, United Kingdom, PLoS Med. 2011 Jul;8(7):e1001057. Epub 2011 Jul 12 (9) Bonnet MGagnidze LGithui WGuerin PJBonte LVaraine FRamsay Performance of LED-Based Fluorescence Microscopy to Diagnose Tuberculosis in a Peripheral Health Centre in Nairobi 2011/01/01 (10) Shenai, S.1; Minion, J.2 et al; International Union Against Tuberculosis and Lung Disease, Volume 15, Number 4, April 2011 pp. 483-488(6) (11) National Consultation on diagnosis and treatment of Pediatric Tuberculosis, LRS Institute of TB and Respiratory Diseaes (12) L Saglam, M Akgun and E Aktas The Journal of International Medical Research: Usefulness of induced sputum and Fibreoptic bronchoscopy specimens in the diagnosis of pulmonary tuberculosis (13)Marcus Conde, Angela Loivos, Valeria Rezende, Sergio Soares, Fernanda Mello, Arthur Reingold, Charles Daley Afranio Kritski: Yield of sputum induction in the diagnosis of pleural tuberculosis (14) HJ Zar, E Tannenbaum, P Apolles, P Roux, D Hanslo and G Hussey: Sputum Induction for the diagnosis of pulmonary tuberculosis in infants and young children in an urban setting in South Africa (Arch Dis Child 2000;82:305-308 doi:10.1136/adc.82.4.305)

APPENDIX -1

CLINICAL PERFORMA

INCLUSION CRITERIA All children up to fifteen years of age who have high probability to have Pulmonary Tuberculosis on clinical and radio logical basis with/without positive Mantouxs test yes/no

All answers should be yes for inclusion

EXCLUSION CRITERIA 1. Children who can produce sputum yes/no 2. Children earlier diagnosed with tuberculosis yes/no 3. Children with wheezing yes/no 4. Children showing signs of upper airway obstruction yes/no 5. Oxygen saturation less than 92% yes/no

All answers should be no for inclusion

Performa

Name: __________________________________________________________________________ Age: ___________________________________________________________________________ Sex: ___________________________________________________________________________ Cr. No.: _________________________________________________________________________ Fathers Name: ___________________________________________________________________ Address: _________________________________________________________________________

_____________________________________________________________________________________

History
Symptoms

Fever

Cough

Breathing difficulty

Weight loss

Anorexia

Hemoptysis

Other

________________________________________________________________________________________ History of Contact: Yes / No If yes, give details: _________________________________________

______________________________________________________________________________________ ______________________________________________________________________________________

EXAMINATION BCG Scar : Yes / No Weight: Pulse rate: / min Respiratory rate: / min

Lymphadenopathy (size & site):

Pallor: Chest finding:

Icterus:

CVS Abdomen: Liver Spleen

Palpable L / N

Ascites

Investigations Date Hb TLC DLC ESR Mx test

Day 1 Gastric Lavage ZN Smear

LED-FM

Induced Sputum

ZN Smear

LED-FM

Day 2 Gastric Lavage ZN Smear

LED-FM

Induced Sputum

ZN Smear

LED-FM

MGIT Culture (Day 1 Sample)

CONSENT FORM

Research study: To evaluate the role of induced sputum in diagnosis of childhood pulmonary tuberculosis. Investigators: Dr. Vikas Kumar Gautam, Post Graduate Student Dr. Varinder Singh , Professor, Department of Pediatrics, LHMC Purpose and Background: The purpose of this study is to study the role of Induced Sputum in bacillary diagnosis of childhood pulmonary tuberculosis. Procedures: If you agree to the inclusion of your child in the study, following procedures will be performed. A. If the child is included in the study, your child will be admitted & sputum sample of child will be sent for investigation. Two methods for collection of sputum will be performed. One is gastric lavage in which child should be empty stomach (over night fast) & early morning sputum sample will be taken from stomach by inserting a tube in to the stomach of the patient from nose. Second one is induced sputum in which patient will inhales nebulised hypertonic saline solution.. B. Benefit: you are free to enroll your child in this study or keep your child away from the study .even if he/she is enrolled in the study, you may withdraw from the study anytime before the procedure is done. He /she will continue to receive the standard medical care and attention that is normally provided to the children with similar illnesses in the hospital. I __________________ (Father/Mother/Guardian) of this child , miss/master __________________ cr no.________ Give my consent to include my child in the above study with the understanding of above details. The aims, methods and benefits of this study have been explained to me in my own language by the doctor. I understand that my decision to include or withdraw my child from the study will not influence the treatment offered to him/her in any way. Date: Signature Name of the child: Relation to the child: