Improving the diagnosis of tuberculosis through optimization of sputum microscopy

Raman Chauhan; Sunil K Raina; Dinesh Kumar; Abhilash Sood

doi:10.4103/AMJM.AMJM_29_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 18 | Issue : 3 | Page : 86-91

Improving the diagnosis of tuberculosis through optimization of sputum microscopy

Raman Chauhan, Sunil K Raina, Dinesh Kumar, Abhilash Sood
Department of Community Medicine, Dr. Rajendra Prasad Government Medical College, Kangra, India; Dr. RadhaKrishan Government Medical College, Hamirpur, Himachal Pradesh, India

Date of Submission	17-Aug-2022
Date of Acceptance	12-Oct-2022
Date of Web Publication	13-Dec-2022

Correspondence Address:
Dr. Sunil K Raina
Department of Community Medicine, Dr. Rajendra Prasad Government Medical College, Kangra, Himachal Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/AMJM.AMJM_29_22

Abstract

Background: Patient compliance is an important component of the case detection. The feasibility of sputum examination on the same day was assessed without compromising on the quality. Materials and Methods: This study was conducted as a cross-sectional study on 215 patients attending the DOTS center in Dr. Rajendra Prasad Government Medical College, Kangra, at Tanda from July 2018 to December 2018. The patients were divided into three arms of the study based on the timing of the sputum sample. Results: There was a high correlation of the test result across all study groups. Combined for all arms, the correlation between the first spot and second spot sputum sample was 0.87, between the first spot and morning sputum sample was 0.82, and between the second spot and morning sputum sample was 0.85. There was no significant statistical difference in the sputum positivity among the groups. The loss to follow-up (LFU) was high (21.9%) for the second and third spot (early morning) samples in group 2, and no LFU was observed for the second spot (after 1 h) and morning sample in group 1 patients. Conclusion: The combination of the first and early morning samples offers a feasible mechanism to diagnose tuberculosis (TB) based on acid fast bacillus identification. Establishing the diagnosis for TB based on two sample results is feasible without any significant drop in the yield.

Keywords: Diagnosis, sputum microscopy, tuberculosis

How to cite this article:
Chauhan R, Raina SK, Kumar D, Sood A. Improving the diagnosis of tuberculosis through optimization of sputum microscopy. Amrita J Med 2022;18:86-91

How to cite this URL:
Chauhan R, Raina SK, Kumar D, Sood A. Improving the diagnosis of tuberculosis through optimization of sputum microscopy. Amrita J Med [serial online] 2022 [cited 2023 Jun 4];18:86-91. Available from: https://ajmonline.org.in/text.asp?2022/18/3/86/363503

Introduction

Tuberculosis (TB) care, including the effective detection and treatment of patients, is central to the global strategy to control the disease. Currently available diagnostics are either less sensitive, time-consuming, or require laboratory infrastructures considerably more advanced than those commonly found in the developing countries where TB burdens are the highest. There is an urgent need for a new TB diagnostic test that is simple, rapid, sensitive, and specific and can be made widely available. The test development pipeline is unlikely to deliver a test with such a profile in the short-to-medium term. The Global Health Diagnostics Forum convened by the Bill and Melinda Gates Foundation has recently developed and utilized a model to evaluate the potential role of better diagnostics in improving TB control in the developing countries.^[1] This modeling exercise has predicted that improving the performance of sputum smear microscopy and reducing the loss of patients during the diagnostic process (e.g., through same-day results) would be associated with considerable public health impact.^[1]

Direct smear microscopy, as currently recommended, is associated with two major problems: low and variable sensitivity and a considerable drop-out of patients during the diagnostic process. A recent study in Cameroon has shown that inadequate examination of smears results in low sensitivity of sputum smear microscopy.^[2]

This is likely to occur where laboratories are overloaded with requests for sputum smear microscopy, as is common in many high-prevalence countries. The patient drop-out during the smear microscopy investigation is also common. A study in Malawi reported 15% of smear-positive cases dropping out of the diagnostic pathway between submitting specimens and being offered treatment.^[3]

Patient compliance is an important component of the case detection. The feasibility of sputum examination on the same day was assessed without compromising on the quality of the results, and this was compared with the current standard sputum collection schedule.

Materials and Methods

This study was conducted as a cross-sectional study on 215 patients attending the DOTS center in Dr. Rajendra Prasad Government Medical College, Kangra, at Tanda from July 2018 to December 2018.

All consecutive patients attending the DOTS center in Dr. Rajendra Prasad Government Medical College, Kangra, at Tanda and fulfilling the inclusion criteria were enrolled for the study. Inclusion criteria for enrolment in the study were cough for ≥2 weeks duration, a clinical suspicion of pulmonary TB, age ≥18 years, and those submitting the informed consent. Exclusion criteria were patients taking anti-tubercular treatment during the last 1 month and those unable to submit the informed consent.

Patients were asked a brief questionnaire to include relevant background, clinical information, and costs associated with travelling to the center and staying overnight. Sputum samples were requested from all patients enrolled. The timing of these samples varied and followed three different arms. Patients coming in the first week of the study were enrolled in arm A, those coming in the second week were enrolled in arm B, and those coming in the third week were enrolled in arm C. This collection scheme was repeated subsequently thereafter till the completion of the study, so as to ensure equal numbers of patients in each arm. All patients submitting for sputum microscopy for a period of 6 months starting from the date of survey were included in the study.

Each patient was instructed to provide sputum samples as per the sputum collection arm he/she was enrolled.

In arm A, three sputum samples were collected as follows:

- One on-the-spot sputum sample at the time of the patient’s first visit;

- A second on-the-spot sample taken 1 h after the first one;

- An early morning sputum sample taken by the patient at home on the day following the initial visit.

In arm B, three sputum samples were collected as follows:

- One on-the-spot sputum sample at the time of the patient’s first visit;

- A second on-the-spot sample taken 2 h after the first one;

- An early morning sputum sample taken by the patient at home on the day following the initial visit.

In arm C (the current standard), two sputum samples were collected as follows:

- One on-the-spot sputum sample at the time of the patient’s first visit,

- An early morning sputum sample taken by the patient at home on the day following the initial visit.

Patients were instructed on the production of good-quality sputum samples. These instructions were given—“Take a very deep breath and hold the air for 5 seconds, then slowly breathe out, thereafter, take another deep breath and cough hard until some sputum comes up into your mouth. Spit the sputum into the plastic cup.” The instructions were similar for patients enrolled each arm except for the timing of the specimen as given above for each respective arm.

Specimens were assessed macroscopically to inspect the quality and quantity and a record made. An appropriate portion of the sputum sample (from the most purulent portion) was collected with an applicator stick or a wire loop and spread on the labeled side of the microscope slide. The slides were air-dried and heat-fixed. All smears were stained by the hot Ziehl-Neelsen technique. All slides were examined blindly by bright-field microscopy at 1000× magnification.

To ensure blinding, an opaque wrap-around sticker was used to cover the laboratory number, and once stained, the slides were mixed before reading. Slides were examined by trained laboratory technicians. Once all slides in the batch had been read, the sticker was removed and the results and time were entered into the study log book.

Results

A total of 215 patients were assessed in three groups without any statistical difference for age across groups. [Table 1] shows that the distribution for age across groups was observed to be without any statistical difference.

Table 1: Frequency distribution and mean age in the three arms of sputum collection

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[Table 2] provides a detailed report of the first, second, and third sputum samples across three arms of sputum collection. On assessment for the first (spot) sputum sample, it is observed that the majority (91.6%) of patients were found to be sputum negative irrespective of groups. The distribution of sputum positivity was found to be statistically similar across all three groups.

Table 2: Results of the first (spot), second, and third (morning) sputum sample across three arms

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In the third arm, only two spot sputum samples were collected. When a comparison was carried out beyond groups 1 and 2, no difference for sputum positivity was found for the second spot results.

On assessment for results of sputum sample collected at the third (early morning) sputum sample, it was observed that no significant difference existed among the groups. This was similar to what was observed for the first and second spot sputum results. In comparison to the first and second spot samples, maximum positive cases (10.7%) were observed in the morning sample.

Also it is clear that the loss to follow-up (LFU) was high (21.9%) for the second third spot (early morning) samples in group 2, and no LFU was observed for the second spot (after 1 h) and morning sample in group 1 patients.

The correlation for sputum results (positive and negative) was done between samples collected at three spots. It was observed that there was a high correlation of test result across all study groups. Combined for all arms, the correlation between the first spot and second spot sputum samples was 0.87, between the first spot and morning sputum sample was 0.82, and between the second spot and morning sputum sample was 0.85.

In the study arm A, there was a high correlation between the second and third sputum samples (0.86) as compared to the first and second or the first and third. For the study arm B, the highest correlation was observed for the first and second spot sputum results (1.0); it was similar (0.83) between the first and third, and the second and third spot sputum results. Since, there were only two spots for the third study arm with observed correlation between the first and second spot for the third arm was observed to be 0.85 [Table 3].

Table 3: Correlation for all tests results in each study arm among different sputum samples for the presence of Mycobacterium tuberculosis

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Discussion

The assessment for TB among 215 chest symptomatic observed expected sputum positive rate of about 10.0% in the present study. The application of three different ways of sputum collection forming three different arms observed high correlation for results of sputum samples for TB. The utility of two samples, as assessed in the third study arm, observed very good correlation of 0.85; it was the second best after correlation (1.0) between the first and second sputum results for the second study arm. Generally, it was observed that the results for acid fast bacillus (AFB) in a sputum sample collected on the day of hospital visit are correlated with sputum collected on the next day morning. Making diagnosis on spot sample and next-day sample will not compromise the yield in terms of case detection based on three samples. The current study hints that the first spot sputum identifies 8.4%, second spot 10.4%, and third spot 11.7% of cases positive for TB.

In the current setting, a combination of the first and early morning samples offers a feasible mechanism to diagnose TB based on AFB identification.

Sputum samples were adequate in amount as generally about 3 mL sample found adequate for AFB testing.^[4] Difficulties observed by the patient in submitting the sputum samples, such as there was a difficulty in submission of the second sputum sample among women due to personal, cultural, socioeconomic, and health care system.^[5] Making diagnostic and treatment practices easy for patients generate ease among patients and improve adherence to program.

AFB detection as a diagnostic method was observed to be the simplest, cheapest, and fastest. The algorithm advised for the diagnosis of TB recommends the examination of three consecutive sputum specimens from TB suspects for the presence of AFB. In the present study, we evaluated the contribution of each specimen to the final detection of TB suspect patients with culture-proven disease. The collection and analysis of retrospective data on patients with culture-proven pulmonary TB, from June 2002 to August 2006 at Dokuz Eylul University Hospital, Turkey, have enabled us to assess the value of examining two sputum specimens in diagnosing this disease. AFB was detected from one or more sputum specimens with direct microscopy in 42% of the cases. An analysis of results of smear examination showed that 97% of AFB were detected from the first specimen and only 3% were obtained from the second smear. The third specimen did not have any additional diagnostic value for the detection of AFB by microscopy suggesting two sputum smears to be sufficient for the early detection of AFB.^[6]

The current international TB guidelines recommend the microscopic examination of three sputum specimens for acid-fast bacilli in the evaluation of persons suspected of having pulmonary TB. A systematic review of studies was conducted to quantify the diagnostic yield of each of three sputum specimens.

By searching multiple databases and sources of 37 eligible studies showed an incremental yield in smear-positive results (in studies using all smear-positive cases as the denominator), and the increase in sensitivity (in studies that used all culture-positive cases as the denominator) of the third specimen was the main outcomes of interest. Although heterogeneity in study methods and results presented challenges for data synthesis, subgroup analyses suggest that the average incremental yield and/or the increase in sensitivity of examining a third specimen ranged between 2% and 5%. Reducing the recommended number of specimens examined from three to two (particularly to two specimens collected on the same day) could benefit TB control programs and potentially increase case detection for several reasons. A number of operational research issues need to be addressed. Studies examining the most effective and efficient means to utilize current technologies for microscopic examination of sputum would be most useful if they followed an internationally coordinated and standardized approach, both to strengthen the country-specific evidence base and to permit comparison among studies.^[7] Retrospective analysis of data of laboratory examined about 4000 specimens with AFB smear and culture. It was confirmed that the first and the second specimens enable M. tuberculosis isolation from a majority of patients (92%), whereas the third or a subsequent specimen collected is of little diagnostic relevance (8%).^[8]

The value of one versus three sputum samples was also assessed in about 7000 patients, where 96.7% of the sputum smear-positive patients had positive smears on the first spot sputum exams as compared to 99.7% who were positive on the morning sputum (P = 0.00). About 93.0% of sputum smear-positive patients had positive smears in all three smears regardless of the smear grading. It was concluded that a single morning smear examination is as sensitive as doing three sputum smear examinations in the diagnosis of sputum smear positive TB.

The incidence of sputum smear positivity differed significantly across age groups, but did not differ between genders.^[9] On contrary, meta-analysis of 23 eligible studies published between 1959 and 2017, involving 8,967 participants who provided 19,252 sputum samples with on-demand spot sputum collection as a main reference standard. It was found that the pooled sputum collection increased TB diagnosis by microscopy (odds ratio [OR]: 1.6, 95% confidence interval (CI): 1.3–1.9, P < 0.0001) or culture (OR: 1.7, 95% CI: 1.2–2.4, P = 0.01).

Providing instructions to the patient before sputum collection, during observed collection, or together with physiotherapy assistance increased diagnostic performance by microscopy (OR: 1.6, 95% CI: 1.3–2.0, P < 0.0001). Collecting early morning sputum did not significantly increase diagnostic performance of microscopy (OR: 1.5, 95% CI: 0.9–2.6, P = 0.2) or culture (OR: 1.4, 95% CI: 0.9–2.4, P = 0.2). Network meta-analysis confirmed these findings and revealed that both pooled and instructed spot sputum collections were similarly effective techniques for increasing the diagnostic performance of microscopy. It was interpreted that the TB diagnoses were substantially increased by either pooled collection or by providing instruction on how to produce a sputum sample taken at any time of the day.^[10]

Reducing the effort of patient can be comprehended by removing the need of the second sputum sample, as it appears to be a redundant practice to identify the AFB. In a study, the need of the second sputum sample observed to be without any improvement.^[11] Same-day sputum samples, if to be used, then it was suggested that they should be assessed by different methods of drawing sample for testing.^[12] Sputum examination for the detection of AFB to diagnose TB is the mainstay for diagnosis in conventional settings. Misclassification of samples may lead to unwarranted discarding of sputum samples that could be used for the diagnosis of TB. There is a need to improve sample collection to reduce the number of saliva samples submitted and the importance of prompt delivery of samples when a preservative is not used.^[13],[14]

Effective training of health workers for sputum induction instructions to the patient observed to be more cost-effective strategy in resource-limited settings.^[15] To examine the effect of patient instruction for provision of sputum samples, a randomized controlled trial involving 174 patients with suspected TB in an urban TB clinic in Indonesia was done. TB was diagnosed in 50.6% of patients who received additional counseling by paramedics compared with 35.5% of patients in the control group following routine diagnostic procedure, corresponding to a 15.1% higher detection rate (95% CI: 2.9–27.4). Significant differences were also found for sputum volume, consistency, positivity rate, and density of acid-fast bacilli.^[16]

In order to increase yield to diagnose patients with TB in settings where a patient perceives barriers to appear for additional samples, the diagnosis based on single sputum sample is also suggested.^[17] Although, the decision to adopt and adapt the strategy to establish diagnosis for TB depends on the validity of the strategy rather than the yield alone. Program managers have their different opinions in their usual settings.

Diagnosis based on a less number of sputum samples have observed with better yield as in a study AFB was detected from 81.0% patients, of which 89.0% were diagnosed from the first and additional 9% (n = 30) were from the second sputum specimen. Validation against gold standard, culture, showed M. tuberculosis growth among 85.0% patients with the first sputum and additional 10.0% were from the second one. The third specimen did not show significant additional diagnostic value for the detection of AFB by microscopy or growth of the M. tuberculosis. The study concluded examining two consecutive sputum specimens is sufficient enough for the effective diagnosis of TB. It can also decrease the laboratory workload and hence improve the quality of work in settings with high TB burden like Bangladesh.^[18] In a study, diagnosis based on the same-day sputum sample observed with as good as collecting two samples.^[19] Again on the contrary, evidence did support the use of early morning sample to establish the diagnosis for TB. The study determined the diagnostic yield from spot and the incremental yield from the early morning (EM) sputum sample cultures among TB-suspected adolescents from rural Uganda. Sputum samples (both spot and early morning) from 1862 adolescents were cultured by the Lowenstein-Jensen (LJ) and Mycobacterium growth indicator tube (MGIT) methods. For spot samples, the diagnostic yields for TB were 19.0% and 57.1% with LJ and MGIT, respectively, whereas the incremental yields (not totals) of the early-morning sample were 9.5% and 42.9% (P < 0.001) with LJ and MGIT, respectively. Among TB-suspected adolescents in rural Uganda, the EM sputum culture has a high incremental diagnostic yield. Therefore, EM sputum in addition to spot sample culture is necessary for improved TB case detection.^[20]

As apprehended in the current study, a collection of two sputum samples at once for the diagnosis of TB in Africa observed with equivalent sensitivity and specificity to the standard initial sample followed by a morning sample strategy.^[21] The utility of the third spot sputum specimen was assessed in Maldova and Uganda. In Moldova, 9% (1141/12525) and in Uganda 20% (7280/36054) of suspects met the TB case definition with at least one positive sputum smear. The incremental yield from the third examination was 4% in Moldova and 3% in Uganda. To detect one additional TB case on a third smear, 273 examinations (95% CI: 200–389) in Moldova and 175 (95% CI: 153–222) in Uganda were thus required. This corresponded to an average of 11 days (8–16) and 7 days (9–12), respectively, to diagnose one additional case of TB. In both countries, the third serial sputum smear examination was inefficient in diagnosing sputum smear-positive TB.^[22]

Limiting the diagnosis based on a limited number of samples depends largely on the quality of implementation of program, which includes the quality of laboratory personnel training for staining and AFB identification. It varies across health facilities or precisely between laboratory technicians.

To conclude, the first spot sputum identifies 8.4% of the cases with TB, the second spot identifies 10.4% of cases with TB, whereas the third spot identifies 11.7% of cases positive for TB. Very good correlation (0.85) was observed between the first and early morning sample for the diagnosis of TB. In the current setting, a combination of the first and early morning samples offers a feasible mechanism to diagnose TB based on AFB identification. Establishing the diagnosis for TB based on two sample results is feasible without any significant drop in the yield.

Financial support and sponsorship

The study has been funded through a research grant from State Task Force on RNTCP.

Conflicts of interest

There are no conflicts of interest.

Authors contribution

RC: data analysis, article writing, and submission; SKR: concept of the study and approval of the article writing; DK: data analysis and interpretation; AS: supervision of data collection.

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