Paucibacillary Specimens Research Articles

Automated Extraction and Amplification for Direct Detection of Mycobacterium tuberculosis Complex in Various Clinical Samples

With the incidence of culture-positive tuberculosis (TB) cases at 25.3 per 100,000 and a 25% rate of TB/HIV coinfection, the TB incidence in French Guiana is the highest of all French regions (3, 5). In this context, there is an urgent need for simple, automated systems for molecular diagnosis of TB that can be adapted to small laboratories. Introduction of a nuclear amplification test in a routine clinical laboratory is an additional expense, and its cost-effectiveness and clinical utility need to be evaluated and seem to be optimized when used for diagnosis in patients with an intermediate-to-high likelihood of TB (8, 14). With these objectives, our study aimed to evaluate a molecular assay developed for the detection of Mycobacterium tuberculosis complex (MTC) on the robotic workstation easyMAG/easyQ (EMEQ; bioMerieux, France), in comparison with culture, the gold standard. Following extraction based on the method described by Boom et al. (1), nucleic acid sequence-based amplification (NASBA) with real-time molecular beacon detection was performed on the extract (9, 12). The specificity of the EMEQ assay was initially evaluated with 33 mycobacterial isolates (16 M. tuberculosis, 2 Mycobacterium bovis, and 15 nontuberculous mycobacterium isolates). Subsequently, detection of MTC was performed on request by specialized physicians during a 1-year prospective study of hospitalized patients (90 clinical specimens split into 25 respiratory and 65 nonrespiratory samples). Samples were processed for acid-fast bacillus (AFB) smear microscopy and cultured, and isolates were identified using biochemical tests in conjunction with restriction fragment length polymorphism of the hsp65 gene (7) and the GenoType mycobacterium assay (Hain Lifescience GmbH, Germany). A total of 52 specimens were culture positive, and 44 were identified as MTC and detected by EMEQ. The 8 remaining culture-positive specimens were atypical mycobacteria (6 Mycobacterium avium and 2 Mycobacterium fortuitum specimens) and found to be negative with EMEQ. No inhibition in the specimens included was detected. Out of 38 specimens that did not grow, 35 were found to be negative with EMEQ (Table 1). Analysis of culture-negative but EMEQ-positive samples resolved 2 out of 3 discrepancies, as follows: (i) for a gastric aspirate specimen, a second aspirate specimen received the same day cultured positive for M. tuberculosis, and (ii) for a urine specimen, another urinary specimen collected 1 month later cultured positive for M. tuberculosis using the Bactec MGIT 960 liquid culture system in another laboratory. Nonetheless, for the single remaining specimen of ascitic fluid, which tested positive by the EMEQ assay in 2 different runs, no further samples could be collected. The patient who provided the remaining specimen, whose ascites was first clinically labeled as being of pancreatic origin, remained “out of sight” despite subsequent calls for specimen collection. Table 1. Results of AFB staining, culture, identification of MTC, and EMEQ for clinical specimens testeda In spite of the reported lack of sensitivity of molecular methods for nonrespiratory and paucibacillary specimens (2, 11, 13), the sensitivity in our study was 100% (Table 2); it was affected by neither the type of specimen nor the AFB counts (9 AFB-negative specimens were EMEQ positive). No false-negative result was observed. The specificity was 100% for laboratory isolates as well as respiratory clinical specimens and 97.1% for nonrespiratory specimens. Table 2. Correlation of EMEQ and MTC by culturea The negative predictive value (NPV) of a diagnostic test for tuberculosis is of great importance in deciding isolation release (6, 14), but predictive values have to be adjusted to the prevalence of the infection in the population tested (4). With a positive predictive value of 97.9% and a NPV of 100% (Table 2), EMEQ appears to be useful as a diagnostic test and to rule out TB in the group studied for intermediate-to-high clinical suspicion. In addition, with 160 min needed for automatic extraction and amplification, incorporation of an internal control enabling the detection of samples inhibitors without duplicate testing of a seeded tube (10), and risk of contamination being minimized by amplification in closed tubes, the EMEQ assay seems to be a reliable method for use in routine clinical laboratories. These preliminary results will need to be confirmed by further studies.

The case of the disappearing mycobacteria in Ziehl–Neelsen-stained smears

A student from Guinea-Bissau presented with a 2-year history of painful swollen lymph nodes in the right side of her neck. Some had opened and formed fistulas that suppurated (Figure 1A). Smears of pus were stained with auramine O (AuO) and screened for mycobacteria at 200 magnification. Very rare images with the morphology of acid-fast organisms were seen in two samples. Following current recommendations, the AuO-stained smears were restained with Ziehl–Neelsen (ZN) to confirm the findings of these images.However, threeexperiencedmicroscopistsobservednoacidfast bacteria, and the ZN-stained smears were reported as negative. Later, Mycobacterium tuberculosis complex grew from all samples. Because ZN-stained mycobacteria fluoresce red–orange when excited with green light, the ZN-stained smears were reexamined with a fluorescence microscope (excitation 546 nm, emission >590 nm). Comparing the same microscopic field, the mycobacteria invisible in transmitted light (Figure 1, top panel: ZN) were clearly visible by fluorescence (Figure 1, bottom panel: ZN-Fl). Because AuO is not excited at 546 nm, smears that are only stained with AuO show no red fluorescence when examined with the filter combination ‘excitation: 546 nm/emission >590 nm’. This case illustrates a disturbing aspect of ZN-stained smears for the diagnosis of tuberculosis. It appears that fluorescence microscopy allows visualization of more mycobacteria, using either AuO or ZN, than does transmitted light microscopy of ZNstained smears. In paucibacillary specimens ZN-stained smears alone may yield false-negative results.

Immune-based diagnostics for TB in children: what is the evidence?

Childhood TB is difficult to diagnose, since disease tends to be paucibacillary and sputum specimens are not easy to obtain in children. Thus, blood-based immune assays are an attractive option. Systematic reviews of serological assays suggest that these tests produce highly inconsistent estimates of sensitivity and specificity, but much of the serology literature is based on adults. In children, there is insufficient evidence to recommend the use of serological tests for active TB diagnosis. Interferon-gamma release assays (IGRA) do not offer substantial improvements in sensitivity over the TST for the diagnosis of active disease. For latent TB infection, the IGRA correlates well with the exposure gradient and seems to have utility in reducing the number of children who undergo preventive therapy due to false-positive TST. Although IGRAs can be used as evidence of TB infection in children, appropriate specimen collection and microbiological confirmation of TB disease should remain a priority.

Performance of Modified Universal Sample Processing Method in a Field Study in Uganda

We developed the universal sample processing (USP) methodology and demonstrated its excellent performance in several validation studies using blinded samples (1-4). An article titled “Poor performance of Universal Sample Processing Method for Diagnosis of Pulmonary Tuberculosis by Smear Microscopy and Culture in Uganda” was published recently in the Journal of Clinical Microbiology (5). In this study, smear sensitivities and specificities were not significantly different in a modified USP protocol that was used and the standard N-acetyl-l-cysteine-NaOH (NALC-NaOH) method. The authors also noted that mycobacterial cultures were more frequently negative in the former method. This in itself is not entirely surprising since it is well known that even established diagnostic techniques, not to mention new and novel methodologies, vary widely in performance from very poor to excellent depending on the study settings. It is most important that new diagnostic techniques are evaluated in well-designed studies, and we thank Cattamanchi and coworkers for carrying out an independent assessment of USP methodology in Uganda (1). However, we wish to highlight some concerns that we have about the misrepresentation of “USP methodology.” While referring to it as the “USP method,” the authors have actually used a modified version of the USP protocol. Based on a careful examination of their findings, we believe that the “poor” performance of the USP method is likely to be a consequence of using a modified USP protocol. Critical aspects of the method were altered. First, bacteria were sedimented at a lower centrifugation speed (3,000 × g instead of the recommended 5,000 to 6,000 × g). This is likely to be a crucial modification since the USP protocol advocates a higher centrifugation speed. Second, the authors do not mention how much of the processed sample was applied; while the NALC-NaOH method advocates the use of only two loopfuls, the USP method prescribes the application of 10% of the processed sample to the slide (2-5). Third, the use of fluorescence microscopy (which is not advocated by USP methodology) likely increased the sensitivity of the NALC-NaOH method without enhancing the detection of acid-fast bacilli on USP slides that are remarkably free of background interference. Fourth, culture sensitivity could be compromised by inefficient bacterial sedimentation, incomplete removal of guanidinium hydrochloride (GuHCl), or the use of 4 to 6 M GuHCl as opposed to the 4 M GuHCl recommended for optimal culture sensitivity. Finally, our previous validation studies were carried out under laboratory conditions, where the finer technicalities of the USP method were strictly adhered to. These stringent procedures might not have been possible in this first field application of the “modified USP” method. The authors mention that the results of the USP smear method were not significantly different from those of the NALC-NaOH method but contradict this statement by defining its performance as “poor.” They also noted that compared to the NALC-NaOH method, the USP method yielded a greater proportion of scanty positive results in support of its stated efficacy in detecting paucibacillary specimens. On this basis and in light of the modifications in the protocol, the title “poor performance” is not justified and the authors should refer to it as “modified USP method.”

Métodos convencionais e moleculares para o diagnóstico da tuberculose pulmonar: um estudo comparativo

To compare four laboratory methods in the diagnosis of pulmonary tuberculosis. Respiratory secretion specimens were collected from 160 patients suspected of having pulmonary tuberculosis. Direct testing for Mycobacterium tuberculosis was carried out using Ziehl-Neelsen and auramine staining. In addition, culture in Löwenstein-Jensen (LJ) medium and polymerase chain reaction (PCR) were used. The strains isolated were identified by means of a radiometric method using p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) and classical methods. The sensitivity of the methods was compared to the gold standard for the diagnosis of pulmonary tuberculosis, based on clinical, radiological and microbiological criteria. Of the 160 patients, 142 were diagnosed with pulmonary tuberculosis according to the gold standard. The sensitivity of Ziehl-Neelsen staining, auramine staining, culture in LJ medium and PCR was 54.2%, 58.4%, 67.6% and 77.5%, respectively, when compared with the diagnostic criterion adopted. All four methods presented 100% specificity. In the identification of mycobacteria, there was high (96.8%) concordance between PCR and the radiometric method using NAP. The sensitivity of PCR was 50.8% in samples with negative sputum smear microscopy results and 98.8% in those with positive results. The sensitivity of PCR was lower in specimens with negative results in sputum smear microscopy and culture than in those with positive results (25.6% and 99.0%, respectively). We found PCR to be a promising method for the diagnosis of pulmonary tuberculosis, even in paucibacillary specimens. Simultaneous identification and faster results are additional advantages of this method.

Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR

BackgroundNucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage.ResultsEighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples.ConclusionWe conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment.

Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial- and host-rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, and blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation ( M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel, efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections.

Advances in Molecular Diagnosis of Tuberculosis.

Acccurate and early diagnosis of tuberculosis is important for its effective management. Several methods for the diagnosis of tuberculosis are available which include tuberculin test, radiological examination and other imaging methods and sputum smear microscopy. Characteristic histopathology is a very important approach but there could be problems to get representative specimen and non specific features could be a problem. The immunology is often not conclusive as antibodies and delayed type hypersensitivity response persist long after the subsidence of sub-clinical or clinical disease. Sputum smear microscopy has both the problems of sensitivity and specificity. Culture is more sensitive and is presently the yardstick for diagnosis, but the time required and frequent negative results in paucibacillary specimens are important limitations. During the last decade, major advances in understanding the genetic structure of mycobacteria have been made. Based on this newer knowledge about the specific gene sequences, several gene probes/gene amplification systems for tuberculosis have been developed [1, 2]. These molecular tools and methods can be used for the confirmation of identity of isolates, direct detection of gene sequences from the clinical specimens and also molecular detection of drug resistance.

Study of gene probes in childhood leprosy.

The objective was to detect nucleic acids of M. leprae in skin lesions of leprosy patients and study the effect of treatment on these nucleic acids, using r-RNA gene probes, using a cross sectional study. The study was carried out at Department of Paediatrics, S.N. Medical College, Agra and Department of Microbiology, Central JALMA Institute for leprosy, Agra. The study included 32 cases of leprosy less than 16 years of age, divided into 3 groups viz. without treatment (12 cases), in middle of treatment (11 cases) and at the end of treatment (9 cases). All cases were subjected to a detailed history and thorough clinical examination. All of them had smear examination and lepromin test done and their skin biopsies were subjected to gene detection. Nucleic acids were isolated from skin biopsies of all cases by standard procedure. After dot blotting of these nucleic acids, they were hybridised with radioactive (p32) r-RNA probes. The results were interpreted after getting the X-ray films processed with background signals from controls. Majority of cases were between 13-16 years of age. As age advanced, the disease moved from tuberculoid end of spectrum towards lepromatous end (p < 0.05). Majority of paucibacillary (PB) cases were lepromin positive while majority of multibacillary (MB) cases were lepromin negative (p < 0.05). In specimens of untreated cases, 50% of PB specimens and 87.5% of MB specimens were positive for r-RNA probes. In multibacillary type 100% smear positive specimens and 67% smear negative specimens were positive for r-RNA probes. In patients during the middle of treatment positivity for r-RNA decreased and 20% of PB specimens and 16.6% MB specimens were positive. At the end of treatment (1 year for PB cases and 2 years for MB cases), the results of r-RNA were negative, which indicated that the treatment regimens used in the study were effective. This study supports the usefulness of r-RNA probes as a diagnostic and therapeutic tool in childhood leprosy.