Microscopic examination and smear negative pulmonary tuberculosis in Ethiopia

Introduction Tuberculosis causes illness among millions of people each year and ranks as the second leading cause of death from infectious disease worldwide. The aim of this study was to investigate the detection rate of microscopic examination and estimate risk of transmission of TB by smear negative pulmonary TB patients. Methods A cross-sectional study and retrospective data analysis on TB were undertaken in Northwest Shewa, Ethiopia. Microscopic examination, bacterial culture and PCR were performed. The statistical analysis was made by using STATA software version 10. Results A total of 92 suspected TB cases was included in the study. Of these, 27.17% (25/92) were positive for microscopic examination and 51% (47/92) for culture. The sensitivity and specificity of microscopic examination with 95% CI were 48.94% (34.08% to 63.93%) and 95.56% (84.82 to 99.33%), respectively. The positive and negative predictive values were 92% (73.93% to 98.78%) and 64.18% (51.53% to 75.53%), respectively. Of 8150 pulmonary TB cases in the retrospective study, 58.9% was smear negative. The proportion of TB-HIV co-infection was 28.66% (96/335). Conclusion The sensitivity of microscopic examination was 48.94% which was very low. The poor sensitivity of this test together with the advent of HIV/AIDS elevated the prevalence of smear negative pulmonary TB. This in turn increased the risk of TB transmission.


Introduction
Tuberculosis (TB) still remains a major global public health problem with 8.6 million incidence and 1.3 million deaths in 2012 [1]. TB causes illness among millions of people each year and ranks as the second leading cause of death from infectious disease worldwide, after the human immunodeficiency virus (HIV) [1]. The majority of people affected by TB are found in economically poor countries where sputum microscopy with a conventional light microscope is the primary method for diagnosing pulmonary TB [2]. In the early 1990s, Multi drug resistant (MDR) -TB strains emerged and have now been found all over the world [3]. Most recently, the global concerns about the emergence of MDR-TB and extensively drug resistant (XDR)-TB have emphasized the need to tackle TB more effectively all over the world [4].
In 2010 / 11, Ethiopia undertook the national population based TB survey which is the first in Africa. The survey report revealed that the prevalence of smear positive TB among adults and all age groups was found to be 108 and 63 per 100, 000 populations, respectively [5]. The prevalence of bacteriologically confirmed TB was found to be 156 per 100,000 populations [5]. Moreover, Ethiopia is one of the 27 high MDR-TB burden countries [1].
Microscopic examination is rapid, relatively simple, inexpensive and highly specific [2]. Laboratory diagnosis of TB is usually done in Ethiopia using acid fast bacilli (AFB) smear microscopic examination [5].
The threshold for detection of AFB in sputum samples under optimal conditions is between 10 4 and 10 5 bacilli per ml [6]. However, the infecting dose of Mycobacterium tuberculosis bacilli is estimated to be fewer than ten organisms [7]. This imply that microscopic examination of AFB identifies the most infectious and misses the less infectious patients. Microscopically missed TB cases are considered to be smear negative. However, patients with smear negative culture positive TB appear to be responsible for about 17% of TB transmission [7]. Therefore, the aim of this study was to investigate the detection rate of microscopic examination of sputum smear and estimate the risk of transmission of TB by smear negative pulmonary TB patients in Ethiopia.

Methods
The study was conducted in Fiche Hospital, North Shewa Zone of Oromia Regional State, Ethiopia. A cross-sectional study design and retrospective data analysis were used. The data were collected in 2007. A total of 92 TB suspected patients were included for the investigation of the detection rate of microscopic examination. In this case, the Suspected TB cases are those with symptoms and signs of suggestive of TB, in particular cough of two weeks or more duration [8]. Furthermore, five years TB case report data of the zone were used to estimate the risk of transmission of TB from microscopically missed smear negative pulmonary TB patients.

Diagnosis of TB
National standard diagnostic algorithm indicated by Figure 1 is used for the diagnosis of TB cases in Ethiopia. Microscopic examination is used for diagnosis, monitoring and defining cure rate of treatment. Three sputum specimens must be collected and examined in two consecutive days (spot-early morning-spot) [5].

Definition of different TB cases
Based on the national guideline, the following TB case classifications were defined [5].
Smear-positive pulmonary TB (PTB+): a patient with at least two initial sputum smear examinations positive for AFB by direct microscopy, or a patient with one initial smear examination positive for AFB by direct microscopy and culture positive, or a patient with one initial smear examination positive for AFB by direct microscope and radiographic abnormalities consistent with active TB as determined by a clinician.
Smear-negative pulmonary TB (PTB-): a patient having symptoms suggestive of TB with at least 3 initial smear examinations negative for AFB by direct microscopy, and 1. No response to a course of broad-spectrum antibiotics, 2. Three negative smear examinations by direct microscopy, 3. Radiological abnormalities consistent with pulmonary TB, 4. Decision by a clinician to treat with a full course of anti-TB or a patient whose diagnosis is based on culture positive for M. tuberculosis but three initial smear examinations negative by direct microscopy.

Sputum collection and transportation
All suspected TB patients in the out patient department (OPD) of Fiche Hospital were invited to participate in the study. Orietation was given to participating individuals regarding the difference between sputum and saliva. They were also told to collect their sputum for two consecutive days, Spot -Early Morning -Spot. The patients were also told to collect their sputum in well aerated places to a volume of 5-10ml during productive cough in separate plastic caps; to tightly close the lid of the cap and disinfect it with tuberculocidal disinfectants. All specimens were kept at 4°C and transported to Aklilu Lemma Institute of Pathobiology, Addis Ababa University for mycobacterial culture using cool ice box.

Microscopic examination of AFB smears
AFB smears were prepared using Ziehl-Neelsen staining technique in Fiche Hospital laboratory. The smears were air dried; flooded with carbol fuchsin; heated gently until steam came out; washed with fine jet of water; decolorized with acid alcohol; counter stained with methylene blue for 1 minute after washing acid alcohol in the same way; air dried (blot dried) and finally examined for minimum of 100 oil immersion fields.

Mycobacterial culture
The sputum specimens were decontaminated by 4% sterile NaOH The data on TB-HIV co-infection were those which were summarized from Fiche Hospital TB Clinic record book.

Ethical consideration
The study was ethically approved by the institutional ethical review board of Aklilu Lemma Institute of Pathobiology, Addis Ababa University. Besides, informed written consent and assent were obtained from each of study participants prior to enrolment.

Data entry and analysis
Statistical analysis was done by STATA software version 10.
Sensitivity, specificity, positive predictive value and negative

Polymerase Chain Reaction (PCR) results
The type of M. tuberculosis complex was identified by mycobacterial culture and confirmed by PCR. In this study, fifteen culture positive specimens were analysed by PCR and 14 of them were confirmed as M. tuberculosis.

Trend of TB distribution
The distribution of smear positive and smear negative pulmonary TB cases in North Shewa zone of Oromia Regional State, Ethiopia

Sputum microscopy is the mainstay of diagnostic methods in
Ethiopia. It is simple, cheap and very fast technique which is highly specific in areas with a very high prevalence of TB [10]. Although it has high specificity, its detection rate is very low.  [16].
Despite of the wide application as primary diagnostic tool, the detection power of microscopic examination is not consistent. This is justified by paucibacillary nature of the disease, quality and quantity of sputum, skill of microscopist, smear preparation, lack of patience and exhaustion of lab technicians due to high load. The other disadvantages of microscopic examination is its inability to diagnose Page number not for citation purposes 5 EPTB, paediatric TB and in individual who is co-infected with HIV-TB [17].
According to National standard diagnostic algorithm (Figure 1), suspected TB cases who are not positive by microscopic examination are followed up for at least 2 to 4 weeks with nonspecific broad-spectrum antibiotics treatment (excluding anti-TB drugs and fluroquinolones) [5].