Experience establishing tuberculosis laboratory capacity in a developing country setting.

OBJECTIVE: To describe the experience of strengthening laboratory diagnosis of tuberculosis (TB) in a resource-limited country with high TB-HIV (human immunodeficiency virus) and multidrug-resistant TB (MDR-TB) prevalence. METHODS: In the Kingdom of Lesotho, which is confronted with high levels of TB, MDR-TB and HIV prevalence, between 2006 and 2008 a coalition of the Foundation for Innovative New Diagnostics, Partners In Health and the World Health Organization renovated the National TB Reference Laboratory and reinforced microscopy services, streamlined conventional culture and drug susceptibility testing (DST) and introduced modern TB diagnostic methods. FINDINGS: It was feasible to establish a biosafety level three facility for solid culture and DST and an external quality assessment programme for smear microscopy within 4 months, all in 2007. Liquid culture and DST were introduced a month later. Preliminary results were comparable to those found in laboratories in industrialised countries. A year later, line-probe assay for the rapid detection of MDR-TB was introduced. DISCUSSION: Through strong political commitment and collaboration, it is possible to rapidly establish quality assured TB diagnostic capacity, including current methods, in a resource-limited setting. Case detection and management for TB and MDR-TB have been greatly enhanced. From a low baseline, TB culture throughput in the laboratory increased ten-fold and has been sustained. This experience has served as a catalyst to translate policy into practice with new diagnostic technologies. It supports global policy setting to enhance and modernise laboratory work in developing countries.

Moxifloxacin and gatifloxacin in an acid model of persistent Mycobacterium tuberculosis.

Studies in the mouse and in humans suggest that use of moxifloxacin and gatifloxacin may shorten the duration of treatment of pulmonary tuberculosis. We describe here the in vitro findings with gatifloxacin and moxifloxacin in regimens similar to those that might be used in the treatment of tuberculosis. The bactericidal activities of moxifloxacin and gatifloxacin were measured alone and in different combinations with isoniazid, rifampicin and pyrazinamide against a 30-day, stationary phase culture, at a pH of 5.9. There was a rapid, irregular fall in colony counts during the first 4 days followed by a slower consistent kill during days 4-21 with a mean kill of -0.36 (SD=2.74) and -0.106 (SD=0.011) log(10)CFU/ml/day, respectively. The 4-21-day kill is considered the best assessment of bactericidal activity against persisting bacilli that prolong treatment. The substitution of either of the quinolones for isoniazid in the control regimen of rifampicin, pyrazinamide and isoniazid did not increase bactericidal activity with log CFU of 5.00 and 4.88, but did result in increased bactericidal action with the log CFU of 4.11 and 4.10 for moxifloxacin and gatifloxacin respectively. Moxifloxacin and gatifloxacin had closely similar activities in all drug combinations. Adding moxifloxacin or gatifloxacin to the control regimen resulted in a significant increase in bactericidal action, considered sufficient to reduce the treatment duration.

Bactericidal action of gatifloxacin, rifampin, and isoniazid on logarithmic- and stationary-phase cultures of Mycobacterium tuberculosis.

The bactericidal activity of gatifloxacin, alone and in combination with isoniazid and rifampin, was studied on both exponential- and stationary-phase cultures of Mycobacterium tuberculosis strain H37Rv. On log-phase cultures, the bactericidal activity of gatifloxacin at 4 microg/ml was rapid and was very similar to that of isoniazid. At concentrations of 0.25 and 4 microg/ml, gatifloxacin enhanced the activity of isoniazid. Killing of the stationary-phase culture was biphasic. During the first 2 days, gatifloxacin at 4 microg/ml slightly increased the limited bactericidal activities of isoniazid and rifampin. However, no further additional bactericidal activity was found during further incubation with isoniazid alone or when gatifloxacin was added to either isoniazid or rifampin. This suggested that the stationary-phase culture contained a mixture of occasionally dividing bacilli that were killed during the first 2 days and true static persisters in the residual population that mimicked those in human lesions. In view of the failure of gatifloxacin to add to the sterilizing activity of isoniazid or rifampin during days 2 to 6 of exposure in the stationary-phase culture, it is unlikely to be a sterilizing drug that can be used to shorten the duration of treatment appreciably when it is added to present treatment regimens.

Action of metronidazole in combination with isoniazid & rifampicin on persisting organisms in experimental murine tuberculosis.

To study the activity of metronidazole on persisting tubercle bacilli, BALB/c mice were infected with Mycobacterium tuberculosis and, after 14 days, treated with isoniazid (H) or rifampicin (R) or isoniazid + rifampicin (HR) for 2 months. An untreated group and a group treated with metronidazole (M) alone served as controls. At the end of 2 months, M was added to the H, R, and HR regimen in half the mice, and the treatment was continued for 1 more month in all mice. At the end of treatment, no viable organisms were detected in the lung or spleen of mice treated with HR or HRM regimens. In contrast, compared to the mice treated with R alone, the log10 colony forming units (cfu) of mice treated with RM were lower by 1.84 and 0.52 in the lung and spleen, respectively. Similarly, compared to the H group, the log10 cfu were lower by 0.67 in the spleen of mice treated with HM, and no additional effect due to M was seen in the lung. Three months after stopping treatment, viable organisms were isolated from both the organs of all the groups. However, the log10 cfu in the lung and spleen for the groups with metronidazole were below the log10 cfu for the respective single or 2 drug groups, except the log10 cfu in the lung for the RM group. These findings suggest that metronidazole, given with bactericidal drugs such as rifampicin and isoniazid may be of value in eliminating persisting tubercle bacilli, but further studies are warranted.

Bactericidal action of ofloxacin, sulbactam-ampicillin, rifampin, and isoniazid on logarithmic- and stationary-phase cultures of Mycobacterium tuberculosis.

The bactericidal actions of ofloxacin and sulbactam-ampicillin, alone and in combination with rifampin and isoniazid, on exponential-phase and stationary-phase cultures of a drug-susceptible isolate of Mycobacterium tuberculosis were studied in vitro. In exponential-phase cultures, all drugs were bactericidal, with the higher concentrations of ofloxacin (5 micrograms/ml) and sulbactam-ampicillin (15 micrograms of ampicillin per ml) being as bactericidal as 1 microgram of isoniazid per ml or 1 microgram of rifampin per ml. In two-drug combinations, both drugs increased the levels of activity of isoniazid and rifampin and were almost as bactericidal as isoniazid-rifampin; they also appeared to increase the level of activity of isoniazid-rifampin in three-drug combinations. In contrast, ofloxacin and sulbactam-ampicillin had little bactericidal activity against stationary-phase cultures and were less active than isoniazid or rifampin alone. Furthermore, in two-drug or three-drug combinations, they did not increase the level of activity of isoniazid, rifampin, or isoniazid-rifampin. These findings suggest that ofloxacin and sulbactam-ampicillin are likely to be most useful in the early stages of treatment and in preventing the emergence of resistance to other drugs but are unlikely to be effective as sterilizing drugs helping to kill persisting lesional bacilli.