Nucleic Acid Amplification Tests for Diagnosis of Smear-Negative TB in a High HIV-Prevalence Setting: A Prospective Cohort Study

Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV.We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment.Of 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28-51) and that of secA1 was 24% (95% CI 15-35). Both tests had specificities of 95% (95% CI 90-98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate.The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations

Utility of nucleic acid amplification techniques for the diagnosis of pulmonary tuberculosis in sub-Saharan Africa.

SETTING: Lusaka, Zambia. OBJECTIVES: To investigate the utility of nucleic amplification tests for the diagnosis of pulmonary tuberculosis in a resource-poor setting with a high incidence of human immunodeficiency virus (HIV). DESIGN: Sputum specimens from suspects attending a referral chest clinic were examined by low-cost 'in-house' one-tube nested polymerase chain reaction (PCR), the enhanced Gen-Probe Amplified Mycobacterium Direct Test (AMTD), auramine smear and Lowenstein-Jensen culture. RESULTS: PCR and AMTD detected respectively 80% and 92% of smear-positive specimens and 40% and 60% of smear-negative, culture-positive specimens. AMTD was positive for 18 culture-negative suspects; subsequent investigation indicated these to be six confirmed tuberculosis patients, nine judged from radiological data and clinical follow-up studies to have pulmonary tuberculosis, and three non-tuberculosis patients. Sensitivity for smear, culture, PCR and AMTD, when compared to a gold standard incorporating both microbiological and clinical data, was respectively 29%, 69%, 55% and 81%. CONCLUSION: In this setting, the sensitivity of the low-cost PCR proved insufficient for its effective use as a tool for diagnosing pulmonary tuberculosis, while AMTD performed considerably better than the current laboratory methods for diagnosis of pulmonary tuberculosis. However, the high cost of this technology may limit its application in the public sector of low-income countries

HIV-1 and recurrence, relapse, and reinfection of tuberculosis after cure: a cohort study in South African mineworkers.

BACKGROUND: The proportion of recurrent tuberculosis cases attributable to relapse or reinfection and the risk factors associated with these different mechanisms are poorly understood. We followed up a cohort of 326 South African mineworkers, who had successfully completed treatment for pulmonary tuberculosis in 1995, to determine the rate and mechanisms of recurrence. METHODS: Patients were examined 3 and 6 months after cure, and then were monitored by the routine tuberculosis surveillance system until December, 1998. IS6110 DNA fingerprints from initial and subsequent episodes of tuberculosis were compared to determine whether recurrence was due to relapse or reinfection All patients gave consent for HIV-1 testing. FINDINGS: During follow-up (median 25.1 months, IQR 13.2-33.4), 65 patients (20%) had a recurrent episode of tuberculosis, a recurrence rate of 10.3 episodes per 100 person-years at risk (PYAR)-16.0 per 100 pyar in HIV-1-positive patients and 6.4 per 100 pyar in HIV-1-negative patients. Paired DNA fingerprints were available in 39 of 65 recurrences: 25 pairs were identical (relapse) and 14 were different (reinfection). 93% (13/14) of recurrences within the first 6 months were attributable to relapse compared with 48% (12/25) of later recurrences. HIV-1 infection was a risk factor for recurrence (hazard ratio 2.4, 95% CI 1.5-4.0), due to its strong association with disease caused by reinfection (18.7 2.4-143), but not relapse (0.58; 0.24-1.4). Residual cavitation and increasing years of employment at the mine were risk factors for relapse. INTERPRETATION: In a setting with a high risk of tuberculous infection, HIV-1 increases the risk of recurrent tuberculosis because of an increased risk of reinfection. Interventions to prevent recurrent disease, such as lifelong chemoprophylaxis in HIV-1-positive tuberculosis patients, should be further assessed

Comparison of two bacteriophage tests and nucleic acid amplification for the diagnosis of pulmonary tuberculosis in sub-Saharan Africa.

SETTING: National reference laboratory in Zambia, a high-incidence setting with a high prevalence of HIV infection. OBJECTIVE: To compare the performance of a commercial bacteriophage kit with a nucleic acid amplification kit and an 'in-house' bacteriophage method for rapid diagnosis of pulmonary tuberculosis (TB). METHODS: Sputum specimens from suspected pulmonary TB cases were examined by direct fluorescence microscopy and culture on Löwenstein Jensen (LJ). In a blinded study, remaining samples were tested by AMTD and FASTPlaqueTB or an in-house bacteriophage assay. Two specimen decontamination protocols were investigated. RESULTS: Microbial contamination of 40.4% was observed when using the FASTPlaqueTB kit specimen preparation protocol. When compared to culture on LJ, the sensitivity of the FASTPlaqueTB test was 20.7%. Implementation of a modified Petroff's decontamination protocol reduced contamination to 5.8% and the FASTPlaqueTB test detected 8/25 (32%) of culture-positive specimens. The sensitivity of AMTD and smear microscopy for these specimens were 64% and 48%, respectively. In a separate experiment the sensitivity of an in-house bacteriophage assay was 45.3% compared to 64.2% for AMTD and 45.3% for direct smear microscopy. CONCLUSIONS: Additional analysis of sputum specimens by bacteriophage assay provided no advantage in this setting. For the rapid diagnosis of TB, AMTD offered improved sensitivity over direct smear microscopy

Tuberculosis treatment failure and drug resistance--same strain or reinfection?

Tuberculosis patients may have Mycobacterium tuberculosis in their sputum at the end of treatment, and may show new drug resistance, due to either inadequate treatment of the original episode or reinfection with a new strain during therapy. In a cohort study of mineworkers with tuberculosis in South Africa, 57 of 438 patients had positive sputum cultures 6 months after recruitment in 1995. Of the 31 patients who initially had fully sensitive strains, 3 developed multidrug resistance (MDR) and 3 single-drug resistance (SDR). Of the 6 who started with SDR, 3 became MDR. HIV infection was not associated with drug resistance at enrollment or 6 months later. We compared pairs of DNA fingerprints from isolates of M. tuberculosis at recruitment and 6 months later in the 48 patients for whom we had both available. In 45, the pairs were identical. In 1 patient, although both isolates were fully sensitive, the later fingerprint had 1 less band (transposition). In 2 pairs, the fingerprint patterns were completely different: one seemed to be the result of laboratory error and the other was a true reinfection with an MDR strain. Despite a high risk of infection, with a moderate proportion of background drug-resistant strains (11% SDR, 6% MDR), reinfection is not a common cause of treatment failure or drug resistance at 6 months

Optimization of housefly larvae production on pig wastes and brewers’ grains for integrated fish and pig farms in the tropics

Lack of appropriate animal waste management methods in many smallholder farms in the tropics often leads to environmental problems, especially in locations with high population density such as urban and peri-urban areas. On farms integrating pig production to fish farming, manure can be turned into a valuable feed source of high quality protein for fish through housefly larvae and contribute to intensify fish production and reduce cost of fish feed. Three experiments were carried to optimize operating conditions for maggot production on animal wastes and industrial byproducts found in Kinshasa, the capital city of the Democratic Republic of Congo (DRC). The comparisons were: (1) production on pure substrates (manure or brewer’s grains) or mixtures with lysine or blood; (2) exposure time to flies for the insemination of the substrates: and (3) dynamics of larvae production. Mixing brewers’ grains with Lysine or manure and/or blood more than doubled the amount of larvae that were harvested. Brewers’ grains are a good source of energy, but are probably deficient in essential amino acids to support the growth of maggots. It also appears that only the first days of laying eggs are important since no difference was observed between temporary and permanent exposure of the substrates to houseflies. The peak of larvae production was reached 6 days after exposure. The addition of cow blood in increasing doses to a mixture of brewers’ grains and manure linearly increased the production of maggots

Nutritive value of tropical forage plants fed to pigs in the Western provinces of the Democratic Republic of the Congo

The nutritive value of 20 forage plants commonly used for feeding pigs in the Democratic Republic of the Congo was studied to determine chemical composition, protein amino acid profiles, mineral content, and in vitro digestibility using a two-step method combining an enzymatic pepsin and pancreatin hydrolysis followed by a 72 h gas-test fermentation. The highest protein contents (270–320 g/kg DM) were obtained for Vigna unguiculata, Psophocarpus scandens, Leucaena leucocephala, Manihot esculenta, and Moringa oleifera. Grasses, Acacia mangium, and Eichhornia crassipes, showed the lowest crude protein (CP) and highest NDF contents. Cajanus cajan and Trypsacum andersonii had the most balanced amino acid profile, being deficient in lysine and slightly deficient in histidine, while Megathyrsus maximus displayed the highest number of essential amino acids deficiencies. High mineral contents were obtained from, in ascending order, with M. oleifera, V. unguiculata, E. crassipes, Ipomea batatas and Amaranthus hybridus. In vitro dry matter digestibility ranged from 0.25 to 0.52, in vitro CP digestibility from 0.23 to 0.80, in vitro energy digestibility from 0.23 to 0.52. M. esculenta, M. oleifera, I. batatas, Mucuna pruriens, V. unguiculata, P. scandens and A. hybridus showed high digestibilities for all nutrients. Gas production during fermentation of the pepsin and pancreatin-indigestible fraction of the plants varied from 42 ml/g DM for A. mangium to 202 ml/g DM for I. batatas (P<0.001). Short-chain fatty acid production during fermentation varied from 157 to 405 mg/g of the pepsin and pancreatin indigestible fraction. It is concluded that some of these species are interesting sources of proteins and minerals with a good digestibility that might be used more economically than concentrate, especially in smallholder production systems, to improve pig feeding, mineral intake and intestinal health in pigs reared in the tropics