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1.  Whole Genome Sequencing Shows a Low Proportion of Tuberculosis Disease Is Attributable to Known Close Contacts in Rural Malawi 
PLoS ONE  2015;10(7):e0132840.
The proportion of tuberculosis attributable to transmission from close contacts is not well known. Comparison of the genome of strains from index patients and prior contacts allows transmission to be confirmed or excluded.
In Karonga District, Malawi, all tuberculosis patients are asked about prior contact with others with tuberculosis. All available strains from culture-positive patients were sequenced. Up to 10 single nucleotide polymorphisms between index patients and their prior contacts were allowed for confirmation, and ≥ 100 for exclusion. The population attributable fraction was estimated from the proportion of confirmed transmissions and the proportion of patients with contacts.
From 1997–2010 there were 1907 new culture-confirmed tuberculosis patients, of whom 32% reported at least one family contact and an additional 11% had at least one other contact; 60% of contacts had smear-positive disease. Among case-contact pairs with sequences available, transmission was confirmed from 38% (62/163) smear-positive prior contacts and 0/17 smear-negative prior contacts. Confirmed transmission was more common in those related to the prior contact (42.4%, 56/132) than in non-relatives (19.4%, 6/31, p = 0.02), and in those with more intense contact, to younger index cases, and in more recent years. The proportion of tuberculosis attributable to known contacts was estimated to be 9.4% overall.
In this population known contacts only explained a small proportion of tuberculosis cases. Even those with a prior family contact with smear positive tuberculosis were more likely to have acquired their infection elsewhere.
PMCID: PMC4504505  PMID: 26181760
2.  Rapid determination of anti-tuberculosis drug resistance from whole-genome sequences 
Genome Medicine  2015;7(1):51.
Mycobacterium tuberculosis drug resistance (DR) challenges effective tuberculosis disease control. Current molecular tests examine limited numbers of mutations, and although whole genome sequencing approaches could fully characterise DR, data complexity has restricted their clinical application. A library (1,325 mutations) predictive of DR for 15 anti-tuberculosis drugs was compiled and validated for 11 of them using genomic-phenotypic data from 792 strains. A rapid online ‘TB-Profiler’ tool was developed to report DR and strain-type profiles directly from raw sequences. Using our DR mutation library, in silico diagnostic accuracy was superior to some commercial diagnostics and alternative databases. The library will facilitate sequence-based drug-susceptibility testing.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0164-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4446134  PMID: 26019726
3.  PhyTB: Phylogenetic tree visualisation and sample positioning for M. tuberculosis 
BMC Bioinformatics  2015;16(1):155.
Phylogenetic-based classification of M. tuberculosis and other bacterial genomes is a core analysis for studying evolutionary hypotheses, disease outbreaks and transmission events. Whole genome sequencing is providing new insights into the genomic variation underlying intra- and inter-strain diversity, thereby assisting with the classification and molecular barcoding of the bacteria. One roadblock to strain investigation is the lack of user-interactive solutions to interrogate and visualise variation within a phylogenetic tree setting.
We have developed a web-based tool called PhyTB ( to assist phylogenetic tree visualisation and identification of M. tuberculosis clade-informative polymorphism. Variant Call Format files can be uploaded to determine a sample position within the tree. A map view summarises the geographical distribution of alleles and strain-types. The utility of the PhyTB is demonstrated on sequence data from 1,601 M. tuberculosis isolates.
PhyTB contextualises M. tuberculosis genomic variation within epidemiological, geographical and phylogenic settings. Further tool utility is possible by incorporating large variants and phenotypic data (e.g. drug-resistance profiles), and an assessment of genotype-phenotype associations. Source code is available to develop similar websites for other organisms (
PMCID: PMC4429496  PMID: 25968323
Tuberculosis; Phylogeny; Sequencing
4.  Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography–mass spectrometry 
•We developed a sample clean-up method to detect tuberculosis from sputum by GC–MS.•Biomarkers recovered: 64–70% (standards solution), and 36–68% (sputum extracts).•Cholesterol removed: 93–98% (standards solution) and 62–92% (sputum extracts).•Less cholesterol in the filtered extracts avoids overloading of the analytical system.•Analyzing large sample batches will need fewer interruptions for system cleaning.
A proof of principle gas chromatography–mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36–68% for PDIMs, allowing some superior detection of the target analytes.
PMCID: PMC4381843  PMID: 25728371
Cholesterol; Mycobacterium tuberculosis; Solid phase extraction; Molecularly imprinted polymers; Thermochemolysis; GC–MS
5.  Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan 
PLoS ONE  2015;10(2):e0117771.
Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.
PMCID: PMC4342168  PMID: 25719196
6.  Regulation of medical diagnostics and medical devices in the East African community partner states 
Medical devices and in vitro diagnostic tests (IVD) are vital components of health delivery systems but access to these important tools is often limited in Africa. The regulation of health commodities by National Regulatory Authorities is intended to ensure their safety and quality whilst ensuring timely access to beneficial new products. Streamlining and harmonizing regulatory processes may reduce delays and unnecessary expense and improve access to new products. Whereas pharmaceutical products are widely regulated less attention has been placed on the regulation of other health products. A study was undertaken to assess regulation of medical diagnostics and medical devices across Partner States of the East African Community (EAC).
Data was collected during October 2012 through desk based review of documents and field research, including face to face interviews with the assistance of a structured questionnaire with closed and open ended questions. Key areas addressed were (i) existence and role of National Regulatory Authorities; (ii) policy and legal framework for regulation; (iii) premarket control; (iv) marketing controls; (v) post-marketing control and vigilance; (vi) country capacity for regulation; (vii) country capacity for evaluation studies for IVD and (viii) priorities and capacity building for harmonization in EAC Partner States.
Control of medical devices and IVDs in EAC Partner States is largely confined to national disease programmes such as tuberculosis, HIV and malaria. National Regulatory Authorities for pharmaceutical products do not have the capacity to regulate medical devices and in some countries laboratory based organisations are mandated to ensure quality of products used. Some activities to evaluate IVDs are performed in research laboratories but post market surveillance is rare. Training in key areas is considered essential to strengthening regulatory capacity for IVDs and other medical devices.
Regulation of medical devices and in vitro diagnostics has been neglected in EAC Partner States. Regulation is weak across the region, and although the majority of States have a legal mandate to regulate medical devices there is limited capacity to do so. Streamlining regulation in the EAC is seen as a positive aspiration with diagnostic tests considered a priority area for harmonisation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12913-014-0524-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4221680  PMID: 25366990
Regulation; IVD; Diagnostic test; Medical device; East African community
7.  Recurrence due to Relapse or Reinfection With Mycobacterium tuberculosis: A Whole-Genome Sequencing Approach in a Large, Population-Based Cohort With a High HIV Infection Prevalence and Active Follow-up 
The Journal of Infectious Diseases  2014;211(7):1154-1163.
Background. Recurrent tuberculosis is a major health burden and may be due to relapse with the original strain or reinfection with a new strain.
Methods. In a population-based study in northern Malawi, patients with tuberculosis diagnosed from 1996 to 2010 were actively followed after the end of treatment. Whole-genome sequencing with approximately 100-fold coverage was performed on all available cultures. Results of IS6110 restriction fragment-length polymorphism analyses were available for cultures performed up to 2008.
Results. Based on our data, a difference of ≤10 single-nucleotide polymorphisms (SNPs) was used to define relapse, and a difference of >100 SNPs was used to define reinfection. There was no evidence of mixed infections among those classified as reinfections. Of 1471 patients, 139 had laboratory-confirmed recurrences: 55 had relapse, and 20 had reinfection; for 64 type of recurrence was unclassified. Almost all relapses occurred in the first 2 years. Human immunodeficiency virus infection was associated with reinfection but not relapse. Relapses were associated with isoniazid resistance, treatment before 2007, and lineage-3 strains. We identified several gene variants associated with relapse. Lineage-2 (Beijing) was overrepresented and lineage-1 underrepresented among the reinfecting strains (P = .004).
Conclusions. While some of the factors determining recurrence depend on the patient and their treatment, differences in the Mycobacterium tuberculosis genome appear to have a role in both relapse and reinfection.
PMCID: PMC4354982  PMID: 25336729
tuberculosis; relapse; reinfection; HIV; whole-genome sequence; RFLP; recurrence
8.  A robust SNP barcode for typing Mycobacterium tuberculosis complex strains 
Nature Communications  2014;5:4812.
Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type.
Genetic variation in Mycobacterium tuberculosis complex (MTBC) bacteria is responsible for differences in factors such as virulence and transmissibility. Here, the authors analyse the genomes of 1,601 MTBC isolates from diverse geographic locations and identify 62 SNPs that may be used to resolve lineages and sublineages of these strains.
PMCID: PMC4166679  PMID: 25176035
9.  Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification 
PLoS ONE  2014;9(8):e103091.
Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39°C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.
PMCID: PMC4138011  PMID: 25118698
10.  PolyTB: A genomic variation map for Mycobacterium tuberculosis 
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool ( to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest.
PMCID: PMC4066953  PMID: 24637013
Mycobacterium tuberculosis; Database; Genomics; Software; Molecular epidemiology; Whole-genome sequencing
11.  Treatment Outcomes of New Tuberculosis Patients Hospitalized in Kampala, Uganda: A Prospective Cohort Study 
PLoS ONE  2014;9(3):e90614.
In most resource limited settings, new tuberculosis (TB) patients are usually treated as outpatients. We sought to investigate the reasons for hospitalisation and the predictors of poor treatment outcomes and mortality in a cohort of hospitalized new TB patients in Kampala, Uganda
Methods and findings
Ninety-six new TB patients hospitalised between 2003 and 2006 were enrolled and followed for two years. Thirty two were HIV-uninfected and 64 were HIV-infected. Among the HIV-uninfected, the commonest reasons for hospitalization were low Karnofsky score (47%) and need for diagnostic evaluation (25%). HIV-infected patients were commonly hospitalized due to low Karnofsky score (72%), concurrent illness (16%) and diagnostic evaluation (14%). Eleven HIV uninfected patients died (mortality rate 19.7 per 100 person-years) while 41 deaths occurred among the HIV-infected patients (mortality rate 46.9 per 100 person years). In all patients an unsuccessful treatment outcome (treatment failure, death during the treatment period or an unknown outcome) was associated with duration of TB symptoms, with the odds of an unsuccessful outcome decreasing with increasing duration. Among HIV-infected patients, an unsuccessful treatment outcome was also associated with male sex (P = 0.004) and age (P = 0.034). Low Karnofsky score (aHR = 8.93, 95% CI 1.88 – 42.40, P = 0.001) was the only factor significantly associated with mortality among the HIV-uninfected. Mortality among the HIV-infected was associated with the composite variable of CD4 and ART use, with patients with baseline CD4 below 200 cells/µL who were not on ART at a greater risk of death than those who were on ART, and low Karnofsky score (aHR = 2.02, 95% CI 1.02 – 4.01, P = 0.045).
Poor health status is a common cause of hospitalisation for new TB patients. Mortality in this study was very high and associated with advanced HIV Disease and no use of ART.
PMCID: PMC3948371  PMID: 24608875
12.  Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing 
PLoS ONE  2013;8(12):e83012.
Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.
Methods and Findings
We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.
Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.
PMCID: PMC3859632  PMID: 24349420
13.  Development and optimization of a gas chromatography/mass spectrometry method for the analysis of thermochemolytic degradation products of phthiocerol dimycocerosate waxes found in Mycobacterium tuberculosis 
RATIONALE: The phthiocerol dimycocerosates (PDIMs) are certain stable and hydrophobic waxes found in the cell membrane of Mycobacterium tuberculosis, bacteria that cause an infectious disease of growing concern worldwide. Previous studies report the analysis of derivatives of the hydrolysed PDIMs from biological samples, following complex extraction and offline derivatization of PDIMs biomarkers, prior to their analysis by gas chromatography/mass spectrometry (GC/MS).
METHODS: We developed and optimized a GC/MS method based on selected ion monitoring (SIM) to detect the derivatives produced via the thermally assisted hydrolysis and methylation (THM) of the PDIMs from the cell membrane of M. tuberculosis. The extraction of PDIMs from culture is simple, and their thermochemolysis is carried out automatically online, thus avoiding the time-consuming derivatization steps of hydrolysis and esterification, usually performed offline.
RESULTS: For standard PDIMs in petroleum ether, our optimized method gave an excellent linearity (R2 = 0.99) at concentrations between 0.172 and 27.5 ng/mL, a good precision (RSD = 11.42 %), and a limit of detection (LOD) of 100 pg/mL. For the PDIMs extracted from dilutions of M. tuberculosis culture, the method gave good linearity (R2 = 0.9685) and an estimated LOD of 400 CFU/mL (CFU = colony forming units) in sterile distilled water.
CONCLUSIONS: A GC/MS(SIM) method is presented for the rapid and quantitative detection of M. tuberculosis, based on the online thermochemolysis of lipidic biomarkers extracted from the bacterial culture. The method has the potential to be applied in human and veterinary clinical laboratories for the rapid diagnosis of tuberculosis in infected biological samples. © 2013 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons, Ltd.
PMCID: PMC3824232  PMID: 24097393
14.  SpolPred: rapid and accurate prediction of Mycobacterium tuberculosis spoligotypes from short genomic sequences 
Bioinformatics  2012;28(22):2991-2993.
Summary: Spoligotyping is a well-established genotyping technique based on the presence of unique DNA sequences in Mycobacterium tuberculosis (Mtb), the causal agent of tuberculosis disease (TB). Although advances in sequencing technologies are leading to whole-genome bacterial characterization, tens of thousands of isolates have been spoligotyped, giving a global view of Mtb strain diversity. To bridge the gap, we have developed SpolPred, a software to predict the spoligotype from raw sequence reads. Our approach is compared with experimentally and de novo assembly determined strain types in a set of 44 Mtb isolates. In silico and experimental results are identical for almost all isolates (39/44). However, SpolPred detected five experimentally false spoligotypes and was more accurate and faster than the assembling strategy. Application of SpolPred to an additional seven isolates with no laboratory data led to types that clustered with identical experimental types in a phylogenetic analysis using single-nucleotide polymorphisms. Our results demonstrate the usefulness of the tool and its role in revealing experimental limitations.
Availability and implementation: SpolPred is written in C and is available from
Supplementary information: Supplementary data are available at Bioinformatics Online.
PMCID: PMC3496340  PMID: 23014632
15.  Detection of Mycobacterium tuberculosis in Sputum by Gas Chromatography-Mass Spectrometry of Methyl Mycocerosates Released by Thermochemolysis 
PLoS ONE  2012;7(3):e32836.
Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test.
PMCID: PMC3293907  PMID: 22403716
16.  Tuberculosis in Sudan: a study of Mycobacterium tuberculosis strain genotype and susceptibility to anti-tuberculosis drugs 
BMC Infectious Diseases  2011;11:219.
Sudan is a large country with a diverse population and history of civil conflict. Poverty levels are high with a gross national income per capita of less than two thousand dollars. The country has a high burden of tuberculosis (TB) with an estimated 50,000 incident cases during 2009, when the estimated prevalence was 209 cases per 100,000 of the population. Few studies have been undertaken on TB in Sudan and the prevalence of drug resistant disease is not known.
In this study Mycobacterium tuberculosis isolates from 235 patients attending three treatment centers in Sudan were screened for susceptibility to isoniazid, rifampicin, ethambutol and streptomycin by the proportion method on Lowenstein Jensen media. 232 isolates were also genotyped by spoligotyping. Demographic details of patients were recorded using a structured questionnaire. Statistical analyses were conducted to examine the associations between drug resistance with risk ratios computed for a set of risk factors (gender, age, case status - new or relapse, geographic origin of the patient, spoligotype, number of people per room, marital status and type of housing).
Multi drug-resistant tuberculosis (MDR-TB), being resistance to at least rifampicin and isoniazid, was found in 5% (95% CI: 2,8) of new cases and 24% (95% CI: 14,34) of previously treated patients. Drug resistance was associated with previous treatment with risk ratios of 3.51 (95% CI: 2.69-4.60; p < 0.001) for resistance to any drug and 5.23 (95% CI: 2.30-11.90; p < 0.001) for MDR-TB. Resistance was also associated with the geographic region of origin of the patient, being most frequently observed in patients from the Northern region and least in the Eastern region with risk ratios of 7.43 (95%CI:3.42,16.18; p: < 0.001) and 14.09 (95%CI:1.80,110.53; p:0.026) for resistance to any drug and MDR-TB. The major genotype observed was of the Central Asia spoligotype family (CAS1_Delhi), representing 49% of the 232 isolates examined.
We conclude that emergence of drug resistant tuberculosis has the potential to be a serious public health problem in Sudan and that strengthened tuberculosis control and improved monitoring of therapy is needed. Further surveillance is required to fully ascertain the extent of the problem.
PMCID: PMC3166935  PMID: 21846389
17.  Propagation of Respiratory Aerosols by the Vuvuzela 
PLoS ONE  2011;6(5):e20086.
Vuvuzelas, the plastic blowing horns used by sports fans, recently achieved international recognition during the FIFA World Cup soccer tournament in South Africa. We hypothesised that vuvuzelas might facilitate the generation and dissemination of respiratory aerosols. To investigate the quantity and size of aerosols emitted when the instrument is played, eight healthy volunteers were asked to blow a vuvuzela. For each individual the concentration of particles in expelled air was measured using a six channel laser particle counter and the duration of blowing and velocity of air leaving the vuvuzela were recorded. To allow comparison with other activities undertaken at sports events each individual was also asked to shout and the measurements were repeated while using a paper cone to confine the exhaled air. Triplicate measurements were taken for each individual. The mean peak particle counts were 658×103 per litre for the vuvuzela and 3.7×103 per litre for shouting, representing a mean log10 difference of 2.20 (95% CI: 2.03,2.36; p<0.001). The majority (>97%) of particles captured from either the vuvuzela or shouting were between 0.5 and 5 microns in diameter. Mean peak airflows recorded for the vuvuzela and shouting were 6.1 and 1.8 litres per second respectively. We conclude that plastic blowing horns (vuvuzelas) have the capacity to propel extremely large numbers of aerosols into the atmosphere of a size able to penetrate the lower lung. Some respiratory pathogens are spread via contaminated aerosols emitted by infected persons. Further investigation is required to assess the potential of the vuvuzela to contribute to the transmission of aerosol borne diseases. We recommend, as a precautionary measure, that people with respiratory infections should be advised not to blow their vuvuzela in enclosed spaces and where there is a risk of infecting others.
PMCID: PMC3100331  PMID: 21629778
18.  The analysis of para-cresol production and tolerance in Clostridium difficile 027 and 012 strains 
BMC Microbiology  2011;11:86.
Clostridium difficile is the major cause of antibiotic associated diarrhoea and in recent years its increased prevalence has been linked to the emergence of hypervirulent clones such as the PCR-ribotype 027. Characteristically, C. difficile infection (CDI) occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora and allow C. difficile to flourish. One of the relatively unique features of C. difficile is its ability to ferment tyrosine to para-cresol via the intermediate para-hydroxyphenylacetate (p-HPA). P-cresol is a phenolic compound with bacteriostatic properties which C. difficile can tolerate and may provide the organism with a competitive advantage over other gut microflora, enabling it to proliferate and cause CDI. It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol.
We show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012). Tyrosine conversion to p-HPA was only observed under certain conditions. We constructed gene inactivation mutants in the hpdBCA operon in strains R20291 and 630Δerm which curtails their ability to produce p-cresol, confirming the role of these genes in p-cresol production. The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production.
C. difficile converts tyrosine to p-cresol, utilising the hpdBCA operon in C. difficile strains 630 and R20291. The hypervirulent strain R20291 exhibits increased production of and tolerance to p-cresol, which may be a contributory factor to the virulence of this strain and other hypervirulent PCR-ribotype 027 strains.
PMCID: PMC3102038  PMID: 21527013
19.  Effectiveness of the Standard WHO Recommended Retreatment Regimen (Category II) for Tuberculosis in Kampala, Uganda: A Prospective Cohort Study 
PLoS Medicine  2011;8(3):e1000427.
Prospective evaluation of the effectiveness of the WHO-recommended standardized retreatment regimen for tuberculosis by Edward Jones-López and colleagues reveals an unacceptable proportion of unsuccessful outcomes.
Each year, 10%–20% of patients with tuberculosis (TB) in low- and middle-income countries present with previously treated TB and are empirically started on a World Health Organization (WHO)-recommended standardized retreatment regimen. The effectiveness of this retreatment regimen has not been systematically evaluated.
Methods and Findings
From July 2003 to January 2007, we enrolled smear-positive, pulmonary TB patients into a prospective cohort to study treatment outcomes and mortality during and after treatment with the standardized retreatment regimen. Median time of follow-up was 21 months (interquartile range 12–33 months). A total of 29/148 (20%) HIV-uninfected and 37/140 (26%) HIV-infected patients had an unsuccessful treatment outcome. In a multiple logistic regression analysis to adjust for confounding, factors associated with an unsuccessful treatment outcome were poor adherence (adjusted odds ratio [aOR] associated with missing half or more of scheduled doses 2.39; 95% confidence interval (CI) 1.10–5.22), HIV infection (2.16; 1.01–4.61), age (aOR for 10-year increase 1.59; 1.13–2.25), and duration of TB symptoms (aOR for 1-month increase 1.12; 1.04–1.20). All patients with multidrug-resistant TB had an unsuccessful treatment outcome. HIV-infected individuals were more likely to die than HIV-uninfected individuals (p<0.0001). Multidrug-resistant TB at enrolment was the only common risk factor for death during follow-up for both HIV-infected (adjusted hazard ratio [aHR] 17.9; 6.0–53.4) and HIV-uninfected (14.7; 4.1–52.2) individuals. Other risk factors for death during follow-up among HIV-infected patients were CD4<50 cells/ml and no antiretroviral treatment (aHR 7.4, compared to patients with CD4≥200; 3.0–18.8) and Karnofsky score <70 (2.1; 1.1–4.1); and among HIV-uninfected patients were poor adherence (missing half or more of doses) (3.5; 1.1–10.6) and duration of TB symptoms (aHR for a 1-month increase 1.9; 1.0–3.5).
The recommended regimen for retreatment TB in Uganda yields an unacceptable proportion of unsuccessful outcomes. There is a need to evaluate new treatment strategies in these patients.
Please see later in the article for the Editors' Summary
Editors' Summary
One-third of the world's population is currently infected with Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB), and 5%–10% of HIV-uninfected individuals will go on to develop disease and become infectious. The risk of progression from infection to disease in HIV infected is much higher. If left untreated, each person with active TB may infect 10 to 15 people every year, reinforcing the public health priority of controlling TB through adequate treatment. Patients with a previous history of TB treatment are a major concern for TB programs throughout the world because these patients are at a much higher risk of harboring a form of TB that is resistant to the drugs most frequently used, resulting in poorer treatment outcomes and significantly complicating current management strategies. More then 1 million people in over 90 countries need to be “re-treated” after failing, interrupting, or relapsing from previous TB treatment.
Every year, 10%–20% of people with TB in low- and middle-income countries are started on a standardized five-drug retreatment regimen as recommended by the World Health Organization (WHO). Yet, unlike treatment regimens for newly diagnosed TB patients, the recommended retreatment regimen (also known as the category II regimen) has never been properly evaluated in randomized clinical trials or prospective cohort studies. Rather, this regimen was recommended by experts before the current situation of widespread drug-resistant TB and HIV infection.
Why Was This Study Done?
WHO surveillance data suggest that the retreatment regimen is successful in about 70% of patients, but retrospective studies that have evaluated the regimen's efficacy showed variable treatment responses with success rates ranging from 26% to 92%. However, these studies have generally only assessed outcomes at the completion of the retreatment regimen, and few have examined the risk of TB recurrence, especially in people who are also infected with HIV and so are more likely to experience TB recurrence—an issue of particular concern in sub-Saharan Africa. Therefore, in this study based in Kampala, Uganda, the researchers conducted a prospective cohort study to assess treatment and survival outcomes in patients previously treated for TB and to identify factors associated with poor outcomes. Given the overwhelming contribution of HIV infection to death, the researchers categorized their survival analysis by HIV status.
What Did the Researchers Do and Find?
The researchers recruited consecutive smear-positive TB patients who were admitted to Mulago Hospital, Kampala, Uganda, for the retreatment of TB with the standard retreatment regimen between July 2003 and January 2007. Eligible patients received daily directly observed therapy and after hospital discharge, were seen every month during their 8-month TB-retreatment course. Home health visitors assessed treatment adherence through treatment card review, monthly pill counts, and patient self-report. After the completion of the retreatment regimen, patients were evaluated for TB recurrence every 3 months for a median of 21 months. The researchers then used a statistical model to identify treatment outcomes and mortality HIV-uninfected and HIV-infected patients.
The researchers found that 29/148 (20%) of HIV-uninfected and 37/140 (26%) of HIV-infected patients had an unsuccessful treatment outcome. Factors associated with an unsuccessful treatment outcome were poor adherence, HIV infection, increasing age, and duration of TB symptoms. All patients with multidrug resistant TB, a form of TB that is resistant to the two most important drugs used to treat TB, had an unsuccessful treatment outcome. In addition, HIV-infected subjects were more likely to die than HIV-uninfected subjects (p<0.0001), and having multidrug resistant TB at enrollment was the only common risk factor for death during follow-up for both HIV-infected and HIV uninfected patients. Other risk factors for death among HIV-infected patients were CD4<50 cells/ml and no antiretroviral therapy treatment and among HIV-uninfected patients were poor adherence and duration of TB symptoms.
What Do These Findings Mean?
The researchers found that although 70%–80% of patients had a successful treatment outcome on completion of antituberculous therapy (a result that compares well with retrospective studies), the standard retreatment regimen had low treatment response rates and was associated with poor long-term outcomes in certain subgroups of patients, particularly those with multidrug resistant TB and HIV.
These findings indicate that the standard retreatment approach to TB as implemented in low- and middle-income settings is inadequate and stress the importance of a new, more effective, strategies. Improved access to rapid diagnostics for TB drug-resistance, second-line TB treatment, and antiretroviral therapy is urgently needed, along with a strong evidence base to guide clinicians and policy makers on how best to use these tools.
Additional Information
Please access these Web sites via the online version of this summary at
The World Health Organization has information on TB, TB retreatment, and multidrug-resistant TB
WHO also provides information on TB/HIV coinfection
The Stop TB Partnership provides information on the global plan to stop TB
PMCID: PMC3058098  PMID: 21423586
20.  Association of Schistosomiasis with False-Positive HIV Test Results in an African Adolescent Population▿  
Journal of Clinical Microbiology  2010;48(5):1570-1577.
This study was designed to investigate the factors associated with the high rate of false-positive test results observed with the 4th-generation Murex HIV Ag/Ab Combination EIA (enzyme immunoassay) within an adolescent and young-adult cohort in northwest Tanzania. (4th-generation assays by definition detect both HIV antigen and antibody.) The clinical and sociodemographic factors associated with false-positive HIV results were analyzed for 6,940 Tanzanian adolescents and young adults. A subsample of 284 Murex assay-negative and 240 false-positive serum samples were analyzed for immunological factors, including IgG antibodies to malaria and schistosoma parasites, heterophile antibodies, and rheumatoid factor (RF) titers. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). False-positive HIV test results were associated with evidence of other infections. False positivity was strongly associated with increasing levels of Schistosoma haematobium worm IgG1, with adolescents with optical densities in the top quartile being at the highest risk (adjusted OR = 40.7, 95% CI = 8.5 to 194.2 compared with the risk for those in the bottom quartile). False positivity was also significantly associated with increasing S. mansoni egg IgG1 titers and RF titers of ≥80 (adjusted OR = 8.2, 95% CI = 2.8 to 24.3). There was a significant negative association between Murex assay false positivity and the levels of S. mansoni worm IgG1 and IgG2 and Plasmodium falciparum IgG1 and IgG4. In Africa, endemic infections may affect the specificities of immunoassays for HIV infection. Caution should be used when the results of 4th-generation HIV test results are interpreted for African adolescent populations.
PMCID: PMC2863920  PMID: 20181896
21.  Molecular Detection of Mixed Infections of Mycobacterium tuberculosis Strains in Sputum Samples from Patients in Karonga District, Malawi ▿ †  
Journal of Clinical Microbiology  2010;48(12):4512-4518.
The occurrence of mixed infections of Mycobacterium tuberculosis is no longer disputed. However, their frequency, and the impact they may have on our understanding of tuberculosis (TB) pathogenesis and epidemiology, remains undetermined. Most previous studies of frequency applied genotyping techniques to cultured M. tuberculosis isolates and found mixed infections to be rare. PCR-based techniques may be more sensitive for detecting multiple M. tuberculosis strains and can be applied to sputum. To date, one study in South Africa has used a PCR approach and suggested that mixed infection could be common. We investigated mixed infections in northern Malawi using two lineage-specific PCR assays targeting the Latin American-Mediterranean (LAM) and Beijing lineages. Compared with spoligotyping, the specificity and sensitivity of both assays was 100%. From 160 culture-positive sputa, mixed LAM and non-LAM strains were detected in 4 sputa belonging to 2 (2.8%) patients. Both patients were HIV positive, with no history of TB. Cultured isolates from both patients showed only LAM by PCR and spoligotyping. In a set of 377 cultured isolates, 4 were mixed LAM and non-LAM. Only one showed evidence of more than one M. tuberculosis strain using IS6110-based restriction fragment length polymorphism (IS6110-RFLP) and spoligotyping analyses. Corresponding sputa for the 4 isolates were unavailable. Mixed Beijing and non-Beijing strains were not detected in this study. Mixed infections appear to be rare in our setting and are unlikely to affect findings based on DNA fingerprinting data. Molecular methods, which avoid the selective nature of culture and target distinct strains, are well suited to detection of mixed infections.
PMCID: PMC3008455  PMID: 20962138
22.  Changes in Mycobacterium tuberculosis Genotype Families Over 20 Years in a Population-Based Study in Northern Malawi 
PLoS ONE  2010;5(8):e12259.
Despite increasing interest in possible differences in virulence and transmissibility between different genotypes of M. tuberculosis, very little is known about how genotypes within a population change over decades, or about relationships to HIV infection.
Methods and Principal Findings
In a population-based study in rural Malawi we have examined smears and cultures from tuberculosis patients over a 20-year period using spoligotyping. Isolates were grouped into spoligotype families and lineages following previously published criteria. Time trends, HIV status, drug resistance and outcome were examined by spoligotype family and lineage. In addition, transmissibility was examined among pairs of cases with known epidemiological contact by assessing the proportion of transmissions confirmed for each lineage, on the basis of IS6110 RFLP similarity of the M tuberculosis strains. 760 spoligotypes were obtained from smears from 518 patients from 1986–2002, and 377 spoligotypes from cultures from 347 patients from 2005–2008. There was good consistency in patients with multiple specimens. Among 781 patients with first episode tuberculosis, the majority (76%) had Lineage 4 (“European/American”) strains; 9% had Lineage 3 (“East-African/Indian”); 8% Lineage 1 (“Indo-Oceanic”); and 2% Lineage 2 (“East-Asian”); others unclassifiable. Over time the proportion of Lineage 4 decreased from >90% to 60%, with an increase in the other 3 lineages (p<0.001). Lineage 1 strains were more common in those with HIV infection, even after adjusting for age, sex and year. There were no associations with drug resistance or outcome, and no differences by lineage in the proportion of pairs in which transmission was confirmed.
This is the first study to describe long term trends in the four M. tuberculosis lineages in a population. Lineage 4 has probably been longstanding in this population, with relatively recent introductions and spread of Lineages1–3, perhaps influenced by the HIV epidemic.
PMCID: PMC2923199  PMID: 20808874
23.  Rate and Amplification of Drug Resistance among Previously-Treated Patients with Tuberculosis in Kampala, Uganda 
Drug-resistant Mycobacterium tuberculosis has emerged as a global threat. In resource-constrained settings, patients with a history of tuberculosis (TB) treatment may have drug-resistant disease and may experience poor outcomes. There is a need to measure the extent of and risk factors for drug resistance in such patients.
From July 2003 through November 2006, we enrolled 410 previously treated patients with TB in Kampala, Uganda. We measured the prevalence of resistance to first- and second-line drugs and analyzed risk factors associated with baseline and acquired drug resistance.
The prevalence of multidrug-resistant TB was 12.7% (95% confidence interval [95% CI], 9.6%–16.3%). Resistance to second-line drugs was low. Factors associated with multidrug-resistant TB at enrollment included a history of treatment failure (odds ratio, 23.6; 95% CI, 7.7–72.4), multiple previous TB episodes (odds ratio, 15.6; 95% CI, 5.0–49.1), and cavities present on chest radiograph (odds ratio, 5.9; 95% CI, 1.2–29.5). Among a cohort of 250 patients, 5.2% (95% CI, 2.8%–8.7%) were infected with M. tuberculosis that developed additional drug resistance. Amplification of drug resistance was associated with existing drug resistance at baseline (P<.01) and delayed sputum culture conversion (P<.01).
The burden of drug resistance in previously treated patients with TB in Uganda is sizeable, and the risk of generating additional drug resistance is significant. There is an urgent need to improve the treatment for such patients in low-income countries.
PMCID: PMC2883442  PMID: 18808360
24.  Field test of a novel detection device for Mycobacterium tuberculosis antigen in cough 
BMC Infectious Diseases  2010;10:161.
Tuberculosis is a highly infectious disease that is spread from person to person by infected aerosols emitted by patients with respiratory forms of the disease. We describe a novel device that utilizes immunosensor and bio-optical technology to detect M. tuberculosis antigen (Ag85B) in cough and demonstrate its use under field conditions during a pilot study in an area of high TB incidence.
The TB Breathalyzer device (Rapid Biosensor Systems Ltd) was field tested in the outpatient clinic of Adama Hospital, Ethiopia. Adults seeking diagnosis for respiratory complaints were tested. Following nebulization with 0.9% saline patients were asked to cough into a disposable collection device where cough aerosols were deposited. Devices were then inserted into a portable instrument to assess whether antigen was present in the sample. Demographic and clinical data were recorded and all patients were subjected to chest radiogram and examination of sputum by Ziehl-Nielsen microscopy. In the absence of culture treatment decisions were based on smear microscopy, chest x-ray and clinical assessment. Breathalyzer testing was undertaken by a separate physician to triage and diagnostic assessment.
Sixty individuals were each subjected to a breathalyzer test. The procedure was well tolerated and for each patient the testing was completed in less than 10 min. Positive breath test results were recorded for 29 (48%) patients. Of 31 patients with a diagnosis of tuberculosis 23 (74%; 95% CI 55-87) were found positive for antigen in their breath and 20 (64%; 95% CI 45-80) were smear positive for acid fast bacilli in their sputum. Six patients provided apparent false positive breathalyzer results that did not correlate with a diagnosis of tuberculosis.
We propose that the breathalyzer device described warrants further investigation as a tool for studying exhalation of M. tuberculosis. The portability, simplicity of use and speed of the test device suggest it may also find use as a tool to aid early identification of infectious cases. We recommend studies be undertaken to determine the diagnostic sensitivity and specificity of the device when compared to microbiological and clinical indicators of tuberculosis disease.
PMCID: PMC2891759  PMID: 20529335
25.  Comparison of rapid tests for detection of rifampicin-resistant Mycobacterium tuberculosis in Kampala, Uganda 
Drug resistant tuberculosis (TB) is a growing concern worldwide. Rapid detection of resistance expedites appropriate intervention to control the disease. Several technologies have recently been reported to detect rifampicin resistant Mycobacterium tuberculosis directly in sputum samples. These include phenotypic culture based methods, tests for gene mutations and tests based on bacteriophage replication. The aim of the present study was to assess the feasibility of implementing technology for rapid detection of rifampicin resistance in a high disease burden setting in Africa.
Sputum specimens from re-treatment TB patients presenting to the Mulago Hospital National TB Treatment Centre in Kampala, Uganda, were examined by conventional methods and simultaneously used in one of the four direct susceptibility tests, namely direct BACTEC 460, Etest, "in-house" phage test, and INNO- Rif.TB. The reference method was the BACTEC 460 indirect culture drug susceptibility testing. Test performance, cost and turn around times were assessed.
In comparison with indirect BACTEC 460, the respective sensitivities and specificities for detecting rifampicin resistance were 100% and 100% for direct BACTEC and the Etest, 94% and 95% for the phage test, and 87% and 87% for the Inno-LiPA assay. Turn around times ranged from an average of 3 days for the INNO-LiPA and phage tests, 8 days for the direct BACTEC 460 and 20 days for the Etest. All methods were faster than the indirect BACTEC 460 which had a mean turn around time of 24 days. The cost per test, including labour ranged from $18.60 to $41.92 (USD).
All four rapid technologies were shown capable of detecting rifampicin resistance directly from sputum. The LiPA proved rapid, but was the most expensive. It was noted, however, that the LiPA test allows sterilization of samples prior to testing thereby reducing the risk of accidental laboratory transmission. In contrast the Etest was low cost, but slow and would be of limited assistance when treating patients. The phage test was the least reproducible test studied with failure rate of 27%. The test preferred by the laboratory personnel, direct BACTEC 460, requires further study to determine its accuracy in real-time treatment decisions in Uganda.
PMCID: PMC2744678  PMID: 19709423

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