The emerging threat of pre-extensively drug-resistant tuberculosis in West Africa: preparing for large-scale tuberculosis research and drug resistance surveillance.

BACKGROUND: Drug-resistant tuberculosis (TB) is a global public health problem. Adequate management requires baseline drug-resistance prevalence data. In West Africa, due to a poor laboratory infrastructure and inadequate capacity, such data are scarce. Therefore, the true extent of drug-resistant TB was hitherto undetermined. In 2008, a new research network, the West African Network of Excellence for Tuberculosis, AIDS and Malaria (WANETAM), was founded, comprising nine study sites from eight West African countries (Burkina Faso, The Gambia, Ghana, Guinea-Bissau, Mali, Nigeria, Senegal and Togo). The goal was to establish Good Clinical Laboratory Practice (GCLP) principles and build capacity in standardised smear microscopy and mycobacterial culture across partnering laboratories to generate the first comprehensive West African drug-resistance data. METHODS: Following GCLP and laboratory training sessions, TB isolates were collected at sentinel referral sites between 2009-2013 and tested for first- and second-line drug resistance. RESULTS: From the analysis of 974 isolates, an unexpectedly high prevalence of multi-drug-resistant (MDR) strains was found in new (6 %) and retreatment patients (35 %) across all sentinel sites, with the highest prevalence amongst retreatment patients in Bamako, Mali (59 %) and the two Nigerian sites in Ibadan and Lagos (39 % and 66 %). In Lagos, MDR is already spreading actively amongst 32 % of new patients. Pre-extensively drug-resistant (pre-XDR) isolates are present in all sites, with Ghana showing the highest proportion (35 % of MDR). In Ghana and Togo, pre-XDR isolates are circulating amongst new patients. CONCLUSIONS: West African drug-resistance prevalence poses a previously underestimated, yet serious public health threat, and our estimates obtained differ significantly from previous World Health Organisation (WHO) estimates. Therefore, our data are reshaping current concepts and are essential in informing WHO and public health strategists to implement urgently needed surveillance and control interventions in West Africa.

Vitamin D as supplementary treatment for tuberculosis: a double-blind, randomized, placebo-controlled trial.

RATIONALE: Vitamin D has been shown to be involved in the host immune response toward Mycobacterium tuberculosis. OBJECTIVES: To test whether vitamin D supplementation of patients with tuberculosis (TB) improved clinical outcome and reduced mortality. METHODS: We conducted a randomized, double-blind, placebo-controlled trial in TB clinics at a demographic surveillance site in Guinea-Bissau. We included 365 adult patients with TB starting antituberculosis treatment; 281 completed the 12-month follow-up. The intervention was 100,000 IU of cholecalciferol or placebo at inclusion and again 5 and 8 months after the start of treatment. MEASUREMENTS AND MAIN RESULTS: The primary outcome was reduction in a clinical severity score (TBscore) for all patients with pulmonary TB. The secondary outcome was 12-month mortality. No serious adverse effects were reported; mild hypercalcemia was rare and present in both arms. Reduction in TBscore and sputum smear conversion rates did not differ among patients treated with vitamin D or placebo. Overall mortality was 15% (54 of 365) at 1 year of follow-up and similar in both arms (30 of 187 for vitamin D treated and 24 of 178 for placebo; relative risk, 1.19 [0.58-1.95]). HIV infection was seen in 36% (131 of 359): 21% (76 of 359) HIV-1, 10% (36 of 359) HIV-2, and 5% (19 of 357) HIV-1+2. CONCLUSIONS: Vitamin D does not improve clinical outcome among patients with TB and the trial showed no overall effect on mortality in patients with TB; it is possible that the dose used was insufficient. Clinical trial registered with www.controlled-trials.com/isrctn (ISRCTN35212132).

Emergence of multidrug-resistant Mycobacterium tuberculosis of the Beijing lineage in Portugal and Guinea-Bissau: a snapshot of moving clones by whole-genome sequencing.

The Beijing genotype comprises a highly disseminated strain type that is frequently associated with multidrug resistant (MDR) tuberculosis (TB) and increased transmissibility but, countries such as Portugal and Guinea-Bissau fall outside the regions phylogeographically associated with this specific genotype. Nevertheless, recent data shows that this genotype might be gradually emerging in these two countries as an underlying cause of primary MDR-TB. Here, we describe the emergence of Mycobacterium tuberculosis Beijing strains associated with MDR-TB in Portugal and Guinea-Bissau demonstrating the presence of the well described superclusters 100-32 and 94-32 in Portugal and Guinea-Bissau, respectively. Genome-wide analysis and comparison with a global genomic dataset of M. tuberculosis Beijing strains, revealed the presence of two genomic clusters encompassing isolates from Portugal and Guinea-Bissau, GC1 (n = 121) and GC2 (n = 39), both of which bore SNP signatures compatible with the 100-32/B0/W148 and 94-32/Central Asia Outbreak clades, respectively. Moreover, GC2 encompasses a cross-border cluster between Portugal, Guinea-Bissau and Brazil thus supporting migration-associated introduction of MDR-TB and subsequent clonal expansion at the community-level. The comparison with global Beijing datasets demonstrates the global reach of the disease and its complex dissemination across multiple countries while in parallel there are clear microevolutionary trajectories towards extensively drug resistant TB.

High mortality risk among individuals assumed to be TB-negative can be predicted using a simple test.

OBJECTIVES: To determine mortality among assumed TB negative (aTBneg) individuals in Guinea-Bissau and to investigate whether plasma levels of soluble urokinase receptor (suPAR) can be used to determine post-consultation mortality risk. METHODS: This prospective West-African cohort study included 1007 aTBneg individuals who were enrolled from 2004 to 2006; 4983 age-matched controls were followed for comparison. Plasma suPAR levels were measured using the suPARnostic ELISA. Survival was analysed using Cox regression, ROC curves and Kaplan-Meier analysis. RESULTS: After 3 months of follow-up, mortality was 21 per 100 person-year-observation (PYO) among aTBneg individuals and three per 100 PYO among the control population [mortality rate ratio (MRR) = 6.92 (95% CI 4.48-10.7)]. SuPAR values ranged between 0.9 and 45 ng/ml in aTBneg individuals. A log-linear relationship was found between suPAR levels <15 ng/ml and mortality. In the log-linear range, a 1 ng/ml increase was associated with a 46% increase in the mortality rate: MRR = 1.46 (95% CI 1.34-1.59). The area under the ROC curves was 0.88 for HIV-positive individuals and 0.79 for HIV-negative individuals. CONCLUSIONS: Our study showed a high mortality rate among aTBneg individuals and demonstrated that suPAR measurements can provide prognostic information on mortality among individuals without disease diagnosis. Measuring suPAR is a technically simple method for determining mortality risk in individuals that are assumed to be TB-negative.

Experimental Evidence for Limited in vivo Virulence of Mycobacterium africanum.

Tuberculosis remains a public health problem and a main cause of death to humans. Both Mycobacterium tuberculosis and Mycobacterium africanum cause tuberculosis. In contrast to M. tuberculosis, which is geographically spread, M. africanum is restricted to West Africa. Differences have also been found in the growth rate and type of disease caused by M. africanum, globally suggesting an attenuation of this bacteria. In this study, we used the mouse model of infection to follow the dynamics of M. africanum infection in terms of bacterial burdens and tissue pathology, as well as the immune response triggered. Our findings support a lower virulence of M. africanum as compared to M. tuberculosis, including in mice lacking IFN-γ, a major protective cytokine in tuberculosis. Furthermore, the lung immune response triggered by M. africanum infection in wild-type animals was characterized by a discrete influx of leukocytes and a modest transcriptional upregulation of inflammatory mediators. Our findings contribute to elucidate the pathogenesis of M. africanum, supporting the hypothesis that this is an attenuated member of the tuberculosis-causing bacteria. Understanding the biology of M. africanum and how it interacts with the host to establish infection will have implications for our knowledge of TB and for the development of novel and better tools to control this devastating disease.

Clonal expansion across the seas as seen through CPLP-TB database: A joint effort in cataloguing Mycobacterium tuberculosis genetic diversity in Portuguese-speaking countries.

Tuberculosis (TB) remains a major health problem within the Community of Portuguese Language Speaking Countries (CPLP). Despite the marked variation in TB incidence across its member-states and continued human migratory flux between countries, a considerable gap in the knowledge on the Mycobacterium tuberculosis population structure and strain circulation between the countries still exists. To address this, we have assembled and analysed the largest CPLP M. tuberculosis molecular and drug susceptibility dataset, comprised by a total of 1447 clinical isolates, including 423 multidrug-resistant isolates, from five CPLP countries. The data herein presented reinforces Latin American and Mediterranean (LAM) strains as the hallmark of M. tuberculosis populational structure in the CPLP coupled with country-specific differential prevalence of minor clades. Moreover, using high-resolution typing by 24-loci MIRU-VNTR, six cross-border genetic clusters were detected, thus supporting recent clonal expansion across the Lusophone space. To make this data available to the scientific community and public health authorities we developed CPLP-TB (available at http://cplp-tb.ff.ulisboa.pt), an online database coupled with web-based tools for exploratory data analysis. As a public health tool, it is expected to contribute to improved knowledge on the M. tuberculosis population structure and strain circulation within the CPLP, thus supporting the risk assessment of strain-specific trends.

CXCL10/IP-10 release is induced by incubation of whole blood from tuberculosis patients with ESAT-6, CFP10 and TB7.7.

IFN-gamma responses to Mycobacterium tuberculosis specific antigens are used as in vitro diagnostic tests for tuberculosis infection. The tests are sensitive and specific for latent and active tuberculosis disease, but sensitivity may be reduced during immunosuppression. The objective of the study was to explore new ways to improve the diagnosis of tuberculosis infection using CXCL10 and IL-2 as alternative markers to IFN-gamma. CXCL10, IL-2, and IFN-gamma responses to stimulation with ESAT-6/CFP10/TB7.7 were assessed in 12 Quantiferon positive, 8 Quantiferon negative tuberculosis patients and 11 Quantiferon negative controls. CXCL10 and IL-2 were determined by multiplex and IFN-gamma by the Quantiferon ELISA. The median antigen specific CXCL10, IFN-gamma, and IL-2 responses in patients with tuberculosis were 870 pg/ml (range 261-1576 pg/ml), 217 pg/ml (81-1273 pg/ml), 59 pg/ml (14-276 pg/ml) respectively, and the CXCL10 responses were significantly higher than any of the other cytokines measured (p=0.001). In 4/7 individuals with a negative (n=6) or indeterminate (n=1) Quantiferon test, antigen specific CXCL10 responses were detectable at high levels ranging from 196-532 pg/ml. In conclusion CXCL10 was strongly induced after M. tuberculosis specific stimulation and sensitivity appeared superior to the Quantiferon test. Our findings suggest that CXCL10 may serve as an alternative or additional marker for the immunodiagnosis of tuberculosis.

Serum 25-hydroxyvitamin D in a West African population of tuberculosis patients and unmatched healthy controls.

BACKGROUND: Little is known regarding vitamin D deficiency (VDD) in African populations and in tuberculosis (TB) patients. VDD has been shown to be associated with TB. OBJECTIVE: We aimed to compare the degree of vitamin D insufficiency (VDI) and VDD in TB patients and healthy adult controls in a West African population. DESIGN: An unmatched case-control study was performed at a Demographic Surveillance Site in Guinea-Bissau. Serum 25-hydroxyvitamin D(3) [25(OH)D(3)] concentrations were measured in 362 TB patients and in 494 controls. RESULTS: Hypovitaminosis D [25(OH)D(3)

Direct Detection by the Xpert MTB/RIF Assay and Characterization of Multi and Poly Drug-Resistant Tuberculosis in Guinea-Bissau, West Africa.

BACKGROUND: This study aimed to evaluate the usefulness of the Xpert MTB/RIF assay for the rapid direct detection of M. tuberculosis complex (MTBC) strains and rifampicin resistance associated mutations in a resource-limited setting such as Guinea-Bissau and its implications in the management of tuberculosis (TB) and drug resistant tuberculosis, complementing the scarce information on resistance and genotypic diversity of MTBC strains in this West African country. METHODS AND RESULTS: This cross-sectional prospective study included 100 consecutive TB patients with positive acid-fast smears at two months of anti-tuberculosis treatment or in a re-treatment situation, between May and December 2012. Resistance to rifampicin was detected using the GeneXpert system and the Xpert MTB/RIF assay. MTBC isolates obtained with the BACTEC MGIT 960 system were tested for susceptibility to first- and second-line anti-tuberculosis drugs. Overall, the prevalence of multidrug-resistant tuberculosis (MDR-TB) was found to be 9 cases. Of these, 67% (6 patients) of confirmed MDR-TB cases had no past history of TB treatment and 33% (3 patients) were previously treated cases. Extensively drug-resistant TB was not found. Molecular typing of the MDR-TB strains revealed recent transmission patterns of imported MDR strains. CONCLUSIONS: The Xpert MTB/RIF assay was reliable for the detection of rifampicin resistant MTBC strains directly from sputum samples of patients undergoing first-line treatment for two months, being more trustworthy than the simple presence of acid-fast bacilli in the smear. Its implementation is technically simple, does not require specialized laboratory infrastructures and is suitable for resource-limited settings when a regular source of electricity and maintenance is available as well as financial and operation sustainability is guaranteed by the health authorities. A high prevalence of MDR-TB among patients at risk of MDR-TB after two months of first-line treatment was found, in support of the WHO recommendations for its use in the management of this risk group.

Community study of the relative impact of HIV-1 and HIV-2 on intrathoracic tuberculosis.

BACKGROUND: HIV-1 infection is associated with an increased incidence of and mortality from tuberculosis. Few community studies have examined the effect of HIV-2 on tuberculosis. METHODS: We investigated the association between HIV-1, HIV-2 and active tuberculosis in four districts (population 42 709) in Bissau, capital of Guinea-Bissau, with the highest known seroprevalence of HIV-2 infection in the world. From May 1996 to June 1998, tuberculosis surveillance and active case finding among contacts was conducted. Patients were HIV-tested, given specific tuberculosis treatment for 8 months and followed regarding mortality. Simultaneously, an HIV sero-survey was performed in a random sample of 1748 permanent residents. RESULTS: During a 25-month period, 366 tuberculosis cases were identified. After excluding cases among visitors to the area, and adjusting for age, the incidence of tuberculosis was 18.3 times higher (95% CI 12.9-26.0) among HIV-1-positive individuals, 13.7 times higher (9.0-20.7) among dually infected (HIV-1 and HIV-2), and 3.0 times higher (2.1-4.3) among HIV-2-infected compared with HIV-negative individuals. HIV-1 and dually infected tuberculosis patients had a higher mortality rate than HIV-negative tuberculosis patients [mortality ratio (MR) 2.68; CI 1.11-6.48 and 2.89; CI 1.13-7.39, respectively]. The survival of HIV-2-positive tuberculosis patients was similar to that of HIV-negative tuberculosis patients (MR 1.19; CI 0.46-3.06). CONCLUSION: The presence of HIV-2 infection increases the incidence of tuberculosis compared with that in non-HIV-infected individuals, but does not affect tuberculosis-related mortality in the short term. In contrast, the presence of HIV-1 infection, alone or with HIV-2, has a several-fold greater impact on both the incidence of and mortality from tuberculosis.

Tuberculosis in Bissau: incidence and risk factors in an urban community in sub-Saharan Africa.

BACKGROUND: Despite the long history of tuberculosis (TB) research, population-based studies from developing countries are rare. METHODS: In a prospective community study in Bissau, the capital of Guinea-Bissau, we assessed the impact of demographic, socioeconomic and cultural risk factors on active TB. A surveillance system in four districts of the capital identified 247 adult (>or=15 years) cases of intrathoracic TB between May 1996 and June 1998. Risk factors were evaluated comparing cases with the 25,189 adults living in the area in May 1997. RESULTS: The incidence of intrathoracic TB in the adult population was 471 per 100 000 person-years. Significant risk factors in a multivariate analysis were increasing age (P < 0.0001), male sex (odds ratio [OR] = 2.58, 95% CI: 1.85, 3.60), ethnic group other than the largest group (Pepel) (OR = 1.64, 95% CI: 1.20, 2.22), adult crowding (OR = 1.68, 95% CI: 1.18, 2.39 for >2 adults in household), and poor quality of housing (OR = 1.66, 95% CI: 1.24, 2.22). Household type was important; adults living alone or with adults of their own sex only, had a higher risk of developing TB than households with husband and wife present, the adjusted OR being 1.76 (95% CI: 1.11, 2.78) for male households and 3.80 (95% CI: 1.69, 8.56) for female households. In a multivariate analysis excluding household type, child crowding was a protective factor, the OR being 0.68 (95% CI: 0.51, 0.90) for households with >2 children per household. CONCLUSIONS: Bissau has a very high incidence of intrathoracic TB. Human immunodeficiency virus (HIV), increasing age, male sex, ethnicity, adult crowding, family structure, and poor housing conditions were independent risk factors for TB. Apart from HIV prevention, TB control programmes need to emphasize risk factors such as socioeconomic inequality, ethnic differences, crowding, and gender.

Utility of the plasma level of suPAR in monitoring risk of mortality during TB treatment.

OBJECTIVE: To investigate whether changes in the plasma level of soluble urokinase plasminogen activator receptor (suPAR) can be used to monitor tuberculosis (TB) treatment efficacy. DESIGN: This prospective cohort study included 278 patients diagnosed with active pulmonary TB and followed throughout the 8-month treatment period. RESULTS: Mortality during treatment was higher in the highest inclusion quartile of suPAR (23%) compared to the lowest three quartiles (7%), the risk ratio being 3.1 (95% CI 1.65-6.07). No association between early smear conversion and subsequent mortality or inclusion suPAR was observed. After 1 and 2 months of treatment, an increase in suPAR compared to at diagnosis was associated with a Mortality Rate Ratio (MRR) of 4.5 (95%CI: 1.45-14.1) and 2.1 (95%CI 0.62-6.82), respectively, for the remaining treatment period. CONCLUSIONS: The present study confirmed that elevated suPAR level at time of initiation of TB treatment is associated with increased risk of mortality. Furthermore, increased suPAR levels after one month of treatment was associated with increased risk of mortality during the remaining 7-month treatment period.

MCP1 SNPs and pulmonary tuberculosis in cohorts from West Africa, the USA and Argentina: lack of association or epistasis with IL12B polymorphisms.

The monocyte chemotactic protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of monocytes to M. tuberculosis infection sites, and previous studies have reported that genetic variants in MCP1 are associated with differential susceptibility to pulmonary tuberculosis (PTB). We examined eight MCP1 single nucleotide polymorphisms (SNPs) in a multi-ethnic, case-control design that included: 321 cases and 346 controls from Guinea-Bissau, 258 cases and 271 controls from The Gambia, 295 cases and 179 controls from the U.S. (African-Americans), and an additional set of 237 cases and 144 controls of European ancestry from the U.S. and Argentina. Two locus interactions were also examined for polymorphisms in MCP1 and interleukin 12B (IL12B), another gene implicated in PTB risk. Examination of previously associated MCP1 SNPs rs1024611 (-2581A/G), rs2857656 (-362G/C) and rs4586 (+900C/T) did not show evidence for association. One interaction between rs2857656 and IL12B SNP rs2288831 was observed among Africans but the effect was in the opposite direction in Guineans (OR = 1.90, p = 0.001) and Gambians (OR = 0.64, p = 0.024). Our data indicate that the effect of genetic variation within MCP1 is not clear cut and additional studies will be needed to elucidate its role in TB susceptibility.

The Guinea-Bissau family of Mycobacterium tuberculosis complex revisited.

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048).

Urine suPAR levels compared with plasma suPAR levels as predictors of post-consultation mortality risk among individuals assumed to be TB-negative: a prospective cohort study.

Plasma levels of the inflammatory biomarker soluble urokinase plasminogen activator (suPAR) have been shown to carry prognostic information in various infectious and inflammatory diseases. The present study aimed to compare the prognostic value of urine suPAR (U-suPAR) to that of plasma suPAR (P-suPAR), thereby exploring the possibility of replacing the blood sample with an easy obtainable urine sample. We enrolled 1,007 adults, older than 15 years of age, with a negative TB diagnosis between April 2004 and December 2006. Levels of U-suPAR and P-suPAR were available in 863 individuals. U-suPAR was measured using a commercial ELISA (suPARnostic®). We found that U-suPAR carried significant prognostic information on mortality for HIV-infected subjects with an area under the ROC curve of 0.75. For HIV-negative individuals, little or no prognostic effect was observed. However, in both HIV positives and negatives, the predictive effect of U-suPAR was found to be inferior to that of P-suPAR.

IP-10, MCP-1, MCP-2, MCP-3, and IL-1RA hold promise as biomarkers for infection with M. tuberculosis in a whole blood based T-cell assay.

BACKGROUND: IFN-gamma responses to M. tuberculosis antigens are used as in-vitro diagnostic tests for tuberculosis infection. The tests are highly specific but sensitivity may be impaired due to immuno-suppression. The objective of this small exploratory study was to compare three novel biomarkers for in-vitro diagnosis of tuberculosis - MCP-1, MCP-3 and IL-1RA - with the current established biomarker IFN-gamma and the newly described IP-10 and MCP-2. METHODS: Whole blood from 8 patents with active tuberculosis and from 7 healthy controls was stimulated with M. tuberculosis specific antigens and mitogen in the Quantiferon In Tube test tubes. Levels of biomarkers were measured using Luminex and ELISA (IFN-gamma). RESULTS: We found all five new biomarkers were expressed in significantly higher concentrations compared to IFN-gamma. IP-10 and MCP-3 levels in the un-stimulated samples were higher in patients compared with controls. CONCLUSION: All biomarkers had diagnostic potential as they could differentiate between the patients and the controls. IP-10 and MCP-2 seemed most promising as they were expressed in high levels with antigen stimulation and were low in the un-stimulated samples. Further studies are needed to explore the potential of these highly expressed novel biomarkers individually and in combination.