M.O. Odubanjo1, and H.O. Dada-Adegbola2

  1. Department of Pathology, University College Hospital, Ibadan.
  2. Department of Medical Microbiology and Parasitology, University College Hospital, Ibadan.


The objective of this study is to audit the processes for the microbiological diagnosis of tuberculosis (TB) in our resource-limited setting. A total of 694 specimens were received from 333 patients. 129 (38.7%) of these patients were positive for TB. 78 (60.5%) were positive on AFB microscopy alone, 13 (10.0%) on culture alone and 38 (29.5%) on both culture and AFB microscopy. Fifty-one (51) cases were positive on culture, 38 of these (74.5%) had growth on Lowensen Jensen culture medium alone, 11 (19.6%) on Pyruvic Acid Enhanced Medium (PAEM) and 3 (5.9%) on both culture media. AFB microscopy showed a diagnostic specificity of 71.6% and a sensitivity of 74.5%. M. Bovis appears to be prevalent and we suggest the need for speciation. If AFB microscopy is to be routinely used alone, without confirmation by culture, then the overriding need is for quality to be fully assured in its use.

Keywords: Mycobacteria, tuberculosis, accuracy, AFB microscopy, culture


Dr. M.O. Odubanjo
Department of Pathology,
University College Hospital,
Ibadan, Nigeria.


Tuberculosis has great political and economic importance both in Nigeria and in the world as a whole. The global burden of the disease is huge.1 The identification of infectious patients is one of the key aspects of tuberculosis control. This is because each individual with untreated smear positive tuberculosis infects 10-15 persons per year.2 A significant proportion of new cases of tuberculosis are sputum smear positive. WHO estimates show that close to 50% of new cases of tuberculosis in 2004 were sputum smear positive.2

Based on the estimated number of incident cases of tuberculosis, Nigeria ranked fifth in the WHO Africa Region in 2006.3 The Tuberculosis Laboratory at the University College Hospital (UCH), the location of this study, has a pivotal role to play in ensuring quality assured bacteriology as part of the WHO Stop TB Strategy 4 in that it is one of the six (6) zonal reference laboratories for Tuberculosis in Nigeria. It serves the South-West zone of Nigeria.3

In Nigeria, while the case notification rate for tuberculosis has become satisfactory, the case detection rate, even within Directly Observed Treatment Short course (DOTS) areas, is still below the target of 70%3,4, hence there is need for a study to audit the diagnostic methods most commonly used.

There are a number of studies from the UCH, Ibadan and other centres in Nigeria describing the peculiarities of tuberculosis in various medical sub-specialties. However, there has been no previous study to audit the diagnostic service for tuberculosis at the UCH. In spite of new technologies5,6,7,8 for diagnosing tuberculosis, AFB microscopy and culture still constitute the mainstay of the diagnostic procedure for tuberculosis at the UCH. The Tuberculosis Laboratory at the UCH, Ibadan has the BACTEC- 460 and MGIT (Mycobacteria Growth Indicator Tube) broth media available with efforts being made to acquire facilities for the molecular diagnosis of tuberculosis.

The global strategy for the control of tuberculosis, DOTS identifies cases by AFB microscopy only,4, 9 this has the advantage of early commencement of treatment. In view of this, it is imperative that AFB microscopy is studied, standardized and its specificity and sensitivity against a more accurate method such as culture determined.

Our aim was to audit the processes for diagnosing tuberculosis in our resource-limited setting with the aim of improving our services and ensuring that the best possible service is rendered to our tuberculosis patients. We hope that this would ultimately improve the case detection rate for tuberculosis at the UCH and other parts of Nigeria.

This was a retrospective study that was carried out in October 2007 recruiting the most recent patients for whom both AFB microscopy and culture had been done, the specimens were examined within the period from January 1996 to December 1998, as routine mycobacterial culture was discontinued shortly after this period. The laboratory records of all suspected cases of tuberculosis for which requests had been received at the Tuberculosis Laboratory of the Department of Medical Microbiology and Parasitology of the UCH, Ibadan were retrieved.

Patient selection
We recruited into the study only those patients who fulfilled the 2006 WHO requirements for the diagnosis of tuberculosis in resource-limited countries.10 This included patients who had at least 2 specimens (sputum, pleural aspirate, bronchial washings) sent for the diagnosis of suspected pulmonary TB and those with at least 1 sample sent for the diagnosis of extrapulmonary TB. By the above WHO specifications,10 patients were considered positive for TB if at least 1 out of 2 or more specimens sent for AFB microscopy was found positive for TB hence, patients who had only 1 sample sent for the diagnosis of suspected pulmonary TB were recruited only if this was positive for TB or from an extrapulmonary site.

The protocol for the diagnosis of pulmonary TB in UCH during the period the samples were obtained was to examine 3 sputum smear specimens, preferably early morning sputum specimens sent on consecutive days. The WHO requirement at the time was for at least 2 of these 3 specimens to be positive for AFB for a diagnosis of pulmonary TB to be made. It has however been shown by several studies that the incremental yield with the 3rd smear is not significant enough to justify the increased workload resulting therefrom, and that 2 pulmonary-related specimens were sufficient for a diagnosis of TB. 11, 12

Patients with multiple specimens sent for the diagnosis of TB had further analysis of the results of only those specimens within the same presentation defined as specimens sent all within the period of 1 week. The demographic data and details about the results of AFB microscopy and culture were obtained and patients with incomplete records were excluded.

Laboratory processes
Specimens from sputum and other non-sterile sites were decontaminated with 5% sodium hypochlorite solution (household bleach, JIK™, trademark Reckitt Benckiser) before centrifugation. Specimens from sterile sites such as cerebrospinal fluid were directly centrifuged and then subjected to Hot Ziehl-Neelsen (ZN) staining with 1% carbolfuchsin solution.13 Centrifugation was done at 3500g with the CRU 5000 model centrifuge manufactured by IEC.

Examination of smears made from diagnostic specimens for acid fast bacilli (AFB) was done by 2 different medical laboratory scientists using binocular microscopes with oil immersion objectives. At least one length of a 20 mm long smear is examined carefully. This corresponds to 100 high power fields (HPF’s) examined at 1000x magnification.11 Positivity on AFB microscopy was recorded as scanty or numerous AFB. These correspond to 1-9 AFB/1 length or 100HPF and >10AFB/1 length or 100HPF respectively. Numerous AFB on microscopy corresponds to 1+, 2+ or 3+ positivity according to the IUATLD and WHO grading scales.11 5% of the slides reviewed weekly were selected in a random but systematic manner for re-checking.

The processed specimens were then cultured directly onto Lowenstein-Jensen (L-J) culture medium6 and Pyruvic Acid Egg Medium (PAEM), a modification of the L-J medium in which glycerol is replaced by pyruvic acid to enhance the growth of M. Bovis13. Incubation was at 37oC for up to 8 weeks. The cultures were checked weekly for the growth of mycobacteria. The internal quality control measures used to confirm that the organism identified is mycobacterium tuberculosis include the reduction of nitrates to nitrites, the production of serpentine cords on smears from in-vitro grown colonies and the lack of pigment production in the dark or in the light.5, 6