|Home | About | Journals | Submit | Contact Us | Français|
Tuberculosis is a major cause of childhood morbidity and mortality in Nigeria. Diagnosis of childhood tuberculosis is a global challenge making early treatment a mirage. We investigated the stool of children for the presence of mycobacteria
Stool samples from children aged 3 days to 3 years who presented for postnatal immunization at a large university-based clinic in Nigeria were subjected to Ziehl-Neelsen staining. Samples with acid-fast bacilli were further processed using mycobacterial culture, spoligotyping and deletion typing.
192 stool samples from different children were collected and processed. Thirty (15.6%) had acid-fast bacilli. Of these, 8/30 had Mycobacterium tuberculosis and 1/30 had M. africanum.
About 5 percent (9/192) of apparently well children had evidence of potentially serious tuberculosis infection. The usefulness of stool specimens for diagnosing pediatric tuberculosis warrants further investigation.
More than 2 billion people, approximately one-third of the global population, are infected with Mycobacterium tuberculosis, the major causative organism of tuberculosis (TB) (1). Mycobacterium africanum and Mycobacterium bovis, also members of the Mycobacterium tuberculosis complex, are much less frequent causes of TB in humans. The incidence and prevalence of pediatric TB varies significantly across the globe, driven largely by the burden of the disease in different countries. About 1 million children under 15 years of age develop TB worldwide annually, representing 11% of all TB cases (2). The majority of these cases occur in low-income countries where the prevalence of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) is high (3). Nigeria currently ranks fourth on the list of TB burdened nations globally (1) and pediatric TB accounts for a substantial proportion of these cases (4). Almost 2 million people per year die as a result of TB, mostly in developing countries like Nigeria, but the mortality in children is often underreported. Despite this, TB is one of the ten leading causes of childhood mortality (5).
Young children and especially newborns are at a high risk when exposed to a contagious source (6). A comprehensive review of the natural history of childhood TB showed that primary infection before 2 years of age frequently progressed to active disease within 12 months (7). As such, pediatric TB is a sentinel event reflecting recent TB transmission from an infectious contact in the community. The number of children with TB in a community is an indirect parameter for assessing the effectiveness of local TB control programme (3).
The diagnosis of pulmonary TB (PTB) in children is challenging (8). Children rarely expectorate adequate amount of sputum and the limitations of using other specimens or techniques such as first morning gastric aspirates (considered the best clinical specimens for young children with suspected pulmonary tuberculosis), nasopharyngeal swab, sputum induction and laryngeal swab are well known. Accordingly, there is a strong imperative to evaluate the diagnostic utility of clinical specimens that are more readily collectable. Some investigators have suggested that stool microscopy and culture for M. tuberculosis may be diagnostic in some children with tuberculosis (9, 10,11, 12), but other investigators have described stool evaluation as “worthless” since non-pathogenic acid fast bacilli (AFB) may be found in normal intestinal contents of adults (13, 14). Following the identification of AFB in stool of apparently well children who were being screened for cryptosporidiosis in our immunization clinic, we designed this study to characterize AFB in the stool of children attending the clinic.
Subjects were consecutive children who presented for immunization at the University of Ibadan Health Services Clinic, Ibadan, Nigeria. Stool samples from the children were evaluated using AFB staining. All the AFB positive stool specimens were evaluated further for the presence of mycobacteria.
The Institutional Review Committee of the University of Ibadan and the University College Hospital, Ibadan, Nigeria approved the study. Oral informed consent was obtained from the parents of the children.
Stool samples were collected from each child into a sterile plastic container and kept in the refrigerator at 4°C prior to processing using Ziehl-Neelsen (ZN) staining. The ZN stain was carried out as earlier described by Shrestha et al. (15).
From the stool samples positive by ZN staining, 2–3g was suspended in 5ml of sterile distilled water, mixed and left for 15 minutes to separate, after which 3ml of the supernatant was processed. Using a sterile centrifuge tube, equal amounts of specimen and activated N-acetyl-L-cysteine (NALC)-Na-OH of 3ml each was added. The content of the tube was mixed until the specimen was liquefied and allowed to stand for 15 minutes. Phosphate buffer was added to 10ml mark on the centrifuge tube and mixed, followed by centrifugation for 15minutes at 3,000 × g. The supernatant was decanted; 2ml of phosphate buffer of pH 6.8 was added to re-suspend the pellet. The suspension was inoculated onto Lowenstein-Jensen slopes with pyruvate and/or glycerol and incubated at 37°C for between 8 and 12 weeks. Isolates were harvested for molecular typing analysis by scrapping the growth from slopes into 200 microliters of sterile distilled water and heating at 80°C for 1 hour.
This was carried out as previously described with minor modifications (16). The direct repeat (DR) region was amplified by PCR with oligonucleotide primers derived from the DR sequence. Twenty-five microliters of the following reaction mixture was used for the PCR: 12.5μl of HotStarTaq Master Mix (Qiagen; this solution provides a final concentration of 1.5 mM MgCl2 and 200μM each deoxynucleoside triphosphate), 2μ of each primer (20 pmol each), 5μl of the suspension of heat-killed cells (approx. 10–50ng), and 3.5μl of distilled water). The mixture was heated for 15 minutes at 96°C and subjected to 30 cycles of 1 minute at 96°C, 1 minute at 55°C, and 30 seconds at 72°C. The amplified product was hybridized to a set of 43 immoblized oligonucleotides, each corresponding to one of the unique spacer DNA sequences within the DR locus. After hybridization, the membrane was washed twice for 10 minutes in 2x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2 PO4 and 1 mM EDTA [Ph 7.7])- 0.5% sodium dodecyl sulfate at 60°C and then incubated in 1:4,000-diluted streptavidin-peroxidase conjugate (Boehringer) for 45 to 60 minutes at 42°C. The membrane was washed twice for 10 minute in 2x SSPE-0.5% sodium dodecyl sulfate at 42°C and rinsed with 2x SSPE for 5 minutes at room temperature. Hybridizing DNA was detected by the enhanced chemiluminescence method (Amersham) and by exposure to x-ray film (Hyperfilm ECL; Amersham) as specified by the manufacturer.
The use of deletion analysis for the typing of M. tuberculosis complex strains has been previously described (17, 18). For this work, deletion typing method previously described by Warren et al. (19) was used. In our analysis, we used primers directed against the RD4 and RD9 loci to generate a deletion profile that would allow speciation of the isolate. The multiplex master mix system from Qiagen was used for the PCRs, with primers previously described by Warren and colleagues (19). The PCR mixture was a multiplex reaction, with each PCR reaction containing 1 μl of DNA template, 5 μl Q-buffer, 12.5 μl multiplex master mix (Qiagen) and 0.5 μl of each primer (50 pmol/μl). The total volume of the reaction was made up to 25 μl with water. The reaction was allowed to run for 15 minutes at 95 °C, followed by 45 cycles at 94 °C for 1 minute, 62 °C for 1 minute and 72 °C for 1 minute. After the last cycle, the samples were incubated at 72 °C for 10 minutes. Products were visualized by electrophoresis on 3% agarose gels.
The positive controls included a known M. bovis isolate (AN5) and a known M. tuberculosis isolate provided by the Medical Research Council, Center for Molecular and Cellular Biology, Stellenbosch University, Cape Town, South Africa, whilst the negative control was water. The resulting gel images were analyzed on the basis of their alignment on the gel (i.e. same band size with either of the controls). The RD9 deletion analysis was done to discriminate M. tuberculosis from other Mycobacterium tuberculosis complex (MTC). Those with a deletion at this region were further investigated with primers targeting the RD4 region and this discriminated M. bovis from the other members of the MTC, primers targeting the RD1 mic and RD2 seal regions were later used to confirm the presence of M. africanum relative to M. pinnipedii and M. microti as described by Warren and colleagues (19).
192 children were recruited into the study, comprising 95 males and 97 females, aged 3 days to 3 years. Thirty children had AFB present in their stool specimens. Mycobacterial culture of stool samples from the thirty children yielded a growth in 9 (30%). Spoligotyping and deletion analysis confirmed these isolates as M. tuberculosis complex (M. tuberculosis = 8, and M. africanum = 1) (Table 1 and Figure 1). The patients with the positive stool mycobacterial cultures included three males and six females with ages ranging from 1 week to 15 months (Table 1).
The diagnosis of TB in pediatric patients is often based on case definitions that incorporate signs and symptoms of TB, suggestive chest radiograph, positive tuberculin skin test and contact with an active TB patient. It is less frequently based on laboratory isolation of M. tuberculosis. Existing algorithms, however, have serious shortcomings and development of reliable and widely applicable algorithms is a high research priority. In this study, we diagnosed M. tuberculosis in 27% (8/30) and M. africanum in 3% (1/30) of children who had AFB-positive stool specimens. Other investigators have diagnosed tuberculosis based on isolation of M. tuberculosis from stool specimens, but in different patient populations. Mwachari et al. (20) cultured M. tuberculosis from the stool of 10 (13%) HIV- infected adults with chronic diarrhea in Kenya. Manatsathit et al. (21) also found M. tuberculosis in the stool of 8 (18%) adult AIDS patients in Thailand. In South Africa, 8% and 5% of 66 children with suspected PTB had stool specimens that were AFB positive and M. tuberculosis culture positive, respectively (9). In that study, AFB were identified only in the stool of children who had PTB that was confirmed with positive gastric aspirates, but stool testing was less sensitive than gastric aspirates overall. Our study is unique because the study population included apparently well children who were brought to the clinic for routine immunization. None of the children had diarrhea, but four had cough although clinical data was not collected prospectively in all children. The yield might be higher in children clinically suspected of having TB.
Some findings of this study provide potentially important epidemiological information. First, M. tuberculosis has been implicated in most cases of TB in children; however, we found a case of M. africanum. This is, to our knowledge, the first published isolation of M. africanum in the stool of a Nigerian child. Cases of TB caused by M. africanum have been previously reported in adults from Nigeria (22, 23) and other African countries (24, 25). In Cameroon, there has been a decline in the prevalence of M. africanum (24); but more cases were recently observed in HIV/AIDS patients in The Gambia (25) and Nigeria (23). Secondly, one of the M. tuberculosis strains cultured in this study (isolate from patient with ID=JC8) has not been previously reported in the SpolDB4 database, which contains a comprehensive listing of the M. tuberculosis strains around the world (Figure 1). This implies the possible circulation of poorly characterized or emerging M. tuberculosis strains in Nigeria.
This study should be interpreted in the context of its limitations. Since we did not have the records of HIV testing in the mothers or children, we are unable to correlate our findings with the HIV-infection status of the subjects. Also, full contact tracing was possible only in some of the children with positive stool tests, seriously constricting our ability to comment on the public health significance of the results. Our data cannot be extrapolated to older children because the oldest subject in this study was 3 years old. Finally, we do not have complete data on the subsequent clinical course of the patients. Despite these limitations, our findings add to the evidence that directed stool studies may be useful in pediatric TB diagnosis.
In conclusion, we have described the isolation of M. tuberculosis from the stool of a significant proportion of apparently well children attending the University of Ibadan Health Services Clinic. We have also described the first isolation of M. africanum from the stool of a Nigerian child. The problematic nature of diagnosing TB disease in this age group justifies further investigation of the diagnostic potential of stool specimens and other readily obtainable specimens, perhaps using more sensitive techniques. The limitations of such testing and the population for which it would be applicable are also fertile areas for clinical and laboratory studies.
The project described was supported by Grant Number D43TW007995 from the Fogarty International Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health.
CONFLICT OF INTEREST: NONE