Policy and programmatic directions for the Lesotho tuberculosis programme: Findings of the national tuberculosis prevalence survey, 2019.

INTRODUCTION: The Kingdom of Lesotho has one of the highest burdens of tuberculosis (TB) in the world. A national TB prevalence survey was conducted to estimate the prevalence of bacteriologically confirmed pulmonary TB disease among those ≥15 years of age in 2019. METHOD: A multistage cluster-based cross-sectional survey where residents ≥15 years in 54 clusters sampled from across the country were eligible to participate. Survey participants were screened using a symptom screen questionnaire and digital chest X-ray (CXR). Respondents who acknowledged cough of any duration, fever, weight loss, night sweats and/or had any CXR abnormality in the lungs were asked to provide two spot sputum specimens. All sputum testing was conducted at the National TB Reference Laboratory (NTRL), where samples underwent Xpert MTB/RIF Ultra (1st sample) and MGIT culture (2nd sample). HIV counselling and testing was offered to all survey participants. TB cases were those with Mycobacterium tuberculosis complex-positive samples with culture; and where culture was not positive, Xpert MTB/RIF Ultra (Xpert Ultra) was positive with a CXR suggestive of active TB and no current or prior history of TB. RESULT: A total of 39,902 individuals were enumerated, and of these, 26,857 (67.3%) were eligible to participate; 21,719 (80.9%) participated in the survey of which 8,599 (40%) were males and 13,120 (60%) were females. All 21,719 (100%) survey participants underwent symptom screening and a total of 21,344 participants (98.3%) had a CXR. Of the 7,584 (34.9%) participants who were eligible for sputum examination, 4,190 (55.2%) were eligible by CXR only, 1,455 (19.2%) by symptom screening, 1,630 by both, and 309 by CXR exemption. A total of 6,780 (89.4%) submitted two sputum specimens, and 311 (4.1%) submitted one sample only. From the 21,719 survey participants, HIV counseling and testing was offered to 17,048, and 3,915 (23.0%) were documented as HIV-positive. The survey identified 132 participants with bacteriologically confirmed pulmonary TB thus providing an estimated prevalence of 581 per 100,000 population (95% CI 466-696) for those ≥15 years in 2019. Using the survey results, TB incidence was re-estimated to be 654 per 100,000 (95% CI 406-959), which was comparable to the 2018 TB incidence rate of 611 per 100,000 (95% CI 395-872) reported by the World Health Organization (WHO). The highest TB burden was found in those ≥55 years and among men. The ratio of prevalence to case notification was estimated at 1.22. TB/HIV coinfection was identified in 39 (29.6%) participants. Out of the 1,825 participants who reported a cough, 50% of these participants, mostly men, did not seek care. Those who sought care predominantly went to the public health facilities. CONCLUSION: The TB prevalence survey results confirmed that burden of TB and TB/HIV coinfection remains very high in Lesotho. Given that TB prevalence remains high, and there is a significant proportion of participants with confirmed TB that did not report TB suggestive symptoms. The National TB Programme will need to update its TB screening and treatment algorithms to achieve the End TB targets. A major focus will need to be placed on finding the "missing cases" i.e., undiagnosed or under-reported TB cases, or ensuring that not only TB symptomatic but also those who do not present with typical TB symptoms are promptly identified to reduce further onward transmission.

Genetic profiling using plasma-derived cell-free DNA in therapy-naïve hepatocellular carcinoma patients: a pilot study.

Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine whether plasma-derived cell-free DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy. Patients and methods: Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was carried out using a panel targeting 46 coding and non-coding genes frequently altered in HCCs. Results: In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) of the patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) of the patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5 cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) of the cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations. Conclusion: In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients.

Effect of miR-21 and miR-30b/c on TRAIL-induced apoptosis in glioma cells.

Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. To define novel pathways that regulate susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in glioma, we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant glioma cells, levels of different miRs are increased, and in particular, miR-30b/c and -21. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. T98G-sensitive cells treated with miR-21 or -30b/c become resistant to TRAIL. Furthermore, we demonstrate that miR-30b/c and miR-21 target respectively the 3' untranslated region of caspase-3 and TAp63 mRNAs, and that those proteins mediate some of the effects of miR-30 and -21 on TRAIL resistance, even in human glioblastoma primary cells and in lung cancer cells. In conclusion, we show that high expression levels of miR-21 and -30b/c are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets for TRAIL resistance in glioma.

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.

In vitro and in vivo activities of new rifamycin derivatives against mycobacterial infections.

Several rifamycin derivatives have been developed during the last 15 years for the treatment of mycobacterial infections. For tuberculosis, rifabutin (RFB) showed strong activity and seemed to be suitable when tuberculosis patients were also treated for their AIDS infection. Rifapentine (RPT) was evaluated in patients with or without AIDS for its intermittent use. It displayed promising activity but must be strengthened in situations, such as AIDS or patients without AIDS but with cavities. Rifalazil (RLZ) has been evaluated in mice but the dosages used were much higher than those tolerated by patients. Regarding Mycobacterium avium infections, RFB showed significant prophylactic activity in humans, RPT displayed some activity in mice and RLZ showed modest activity in mice.

Fixed dose combinations for tuberculosis: Lessons learned from clinical, formulation and regulatory perspective.

Worldwide, tuberculosis (TB) remains one of the most important communicable diseases in terms of morbidity and mortality. Its control requires multi-drug therapy for at least six months, which could lead to patient non-compliance, failure of therapy and ultimately resulting in the emergence of drug resistance. Fixed dose combinations (FDCs) in TB therapy reduce the number of tablets to be consumed and thereby increase patient compliance with recommended treatment regimens. Thus, FDCs play a significant role in preventing the emergence of drug resistance and successful treatment. However, the quality of FDCs with respect to variable bioavailability and their registration requirements are major hurdles to their implementation in national TB control programs. It is anticipated that a large global market for FDCs will encourage large-scale production and increased competition, which in turn will result in FDCs at affordable prices. The Global Drug Facility (GDF), established by the World Health Organization (WHO), aims to ensure universal uninterrupted access to quality TB drugs for implementation of directly observed treatment short-course (DOTS) in resource-poor countries. In this program, four FDCs were accepted as the drugs of first choice because of their obvious advantages in controlling TB. This demands the necessity of addressing quality and registration requirements of FDCs systematically. In light of this current knowledge on anti-TB FDCs, their dosage, combinations, available clinical studies and the experiences with TB therapy has been discussed in this article, which should serve as lessons for selection of appropriate FDCs for other diseases such as malaria and AIDS.

Effectiveness of once-weekly rifapentine and moxifloxacin regimens against Mycobacterium tuberculosis in mice.

Mice infected with 1.6 x 10(7) CFU of Mycobacterium tuberculosis were treated 14 days later for 6 months with a regimen of once-weekly 10 mg of rifapentine and 75 mg of isoniazid per kg of body weight supplemented with either 150 mg of streptomycin per kg or 100 mg of moxifloxacin per kg during either both the 2-week daily initial and once-weekly continuation phases or only in the daily 2-week initial phase. On completion of treatment, all lung cultures were negative, except for three mice, each with a single colony: two whose rifapentine-isoniazid regimen was supplemented with streptomycin during the whole course of therapy and one whose rifapentine-isoniazid regimen had no initial daily phase, but was supplemented with streptomycin and moxifloxacin during the whole course of therapy. After 3 months of follow-up, positive lung cultures were obtained from 61 and 56% of mice supplemented with streptomycin during either the full course of therapy or only the daily 2-week initial phase, respectively, and 15 and 50% of mice supplemented with moxifloxacin during either the full course of therapy or only the daily 2-week initial phase, respectively. These results suggest that moxifloxacin has sterilizing activity against M. tuberculosis.

Effectiveness of rifampin, rifabutin, and rifapentine for preventive therapy of tuberculosis in mice.

To identify alternative regimens for preventive therapy of tuberculosis, the pharmacokinetics and antimicrobial activities of rifampin (RMP), rifabutin (RBT), and rifapentine (RPT) were compared in BCG-vaccinated and M. tuberculosis-infected immunocompetent mice. RPT showed the highest serum peak level (Cmax) and the longest half-life (t1/2), whereas RBT displayed the lowest Cmax and the shortest t1/2. On weight-to-weight basis, both RPT and RBT were more bactericidal than RMP. The activity of RMP was significantly reduced when the frequency of administration was reduced from six to three times weekly, whereas significant bactericidal activity was still observed in mice treated with RPT, 10 mg/kg up to once fortnightly, or RBT, 10 mg/kg twice weekly. Because the bactericidal activity of RBT, 10 mg/kg six times/wk for 6 wk, or RPT, 10 mg/kg two times/wk for 12 wk, was comparable to that of RMP, 10 mg/kg six times/wk for 12 wk in mice, the two regimens are appropriate for clinical trials of preventive therapy of tuberculosis.

Fixed dose combination short course chemotherapy in the treatment of pulmonary tuberculosis.

In this study two highly effective chemotherapeutic regimens of 6 and 8 months duration were studied and compared with the standard 12 month therapy under full supervision at the National TB Centre and St. Peter's TB Hospital in Addis Abeba. Patients with direct smear positive pulmonary tuberculosis were admitted to hospital during the initial phase of treatment for two months. At two months, sputum conversion rates, by direct smear and culture respectively, were 82% and 80% on Rifater, and 86 or 88% and 86% on the regimens containing separate preparations of isoniazid rifampicin and pyrazinamide, compared to 60% and 30% on the standard regimen. It was concluded that short course regimens of six or eight months with drugs either in combined form or separate preparations are more effective than the standard regimen.