Mapping of Mycobacterium tuberculosis Complex Genetic Diversity Profiles in Tanzania and Other African Countries

The aim of this study was to assess and characterize Mycobacterium tuberculosis complex (MTBC) genotypic diversity in Tanzania, as well as in neighbouring East and other several African countries. We used spoligotyping to identify a total of 293 M. tuberculosis clinical isolates (one isolate per patient) collected in the Bunda, Dar es Salaam, Ngorongoro and Serengeti areas in Tanzania. The results were compared with results in the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Genotyping and phylogeographical analyses highlighted the predominance of the CAS, T, EAI, and LAM MTBC lineages in Tanzania. The three most frequent Spoligotype International Types (SITs) were: SIT21/CAS1-Kili (n = 76; 25.94%), SIT59/LAM11-ZWE (n = 22; 7.51%), and SIT126/EAI5 tentatively reclassified as EAI3-TZA (n = 18; 6.14%). Furthermore, three SITs were newly created in this study (SIT4056/EAI5 n = 2, SIT4057/T1 n = 1, and SIT4058/EAI5 n = 1). We noted that the East-African-Indian (EAI) lineage was more predominant in Bunda, the Manu lineage was more common among strains isolated in Ngorongoro, and the Central-Asian (CAS) lineage was more predominant in Dar es Salaam (p-value<0.0001). No statistically significant differences were noted when comparing HIV status of patients vs. major lineages (p-value = 0.103). However, when grouping lineages as Principal Genetic Groups (PGG), we noticed that PGG2/3 group (Haarlem, LAM, S, T, and X) was more associated with HIV-positive patients as compared to PGG1 group (Beijing, CAS, EAI, and Manu) (p-value = 0.03). This study provided mapping of MTBC genetic diversity in Tanzania (containing information on isolates from different cities) and neighbouring East African and other several African countries highlighting differences as regards to MTBC genotypic distribution between Tanzania and other African countries. This work also allowed underlining of spoligotyping patterns tentatively grouped within the newly designated EAI3-TZA lineage (remarkable by absence of spacers 2 and 3, and represented by SIT126) which seems to be specific to Tanzania. However, further genotyping information would be needed to confirm this specificity

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

Genomic and functional analyses of mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance.

CAPRISA, 2016.Abstract available in PDF file

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

11 páginas, 2 figuras, 1 tablaInappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)Peer reviewe

Digital PCR to detect and quantify heteroresistance in drug resistant Mycobacterium tuberculosis.

Drug resistance in Mycobacterium tuberculosis presents an enormous public health threat. It is typically defined as >1% of drug resistant colonies using the agar proportion method. Detecting small numbers of drug resistant Tb in a population, also known as heteroresistance, is challenging with current methodologies. Here we have utilized digital PCR to detect heteroresistance within M. tuberculosis populations with excellent accuracy versus the agar proportion method. We designed dual TaqMan-MGB probes to detect wild-type and mutant sequences of katG (315), rpoB (531), gyrA (94,95) and rrs (1401), genes that associate with resistance to isoniazid, rifampin, fluoroquinolone, and aminoglycoside respectively. We generated heteroresistant mixtures of susceptible and extensively drug resistant Tb, followed by DNA extraction and digital PCR. Digital PCR yielded a close approximation to agar proportion's percentages of resistant colonies, and yielded 100% concordance with agar proportion's susceptible/resistant results. Indeed, the digital PCR method was able to identify mutant sequence in mixtures containing as little as 1000∶1 susceptible:resistant Tb. By contrast, real-time PCR or PCR followed by Sanger sequencing were less sensitive and had little resolution to detect heteroresistance, requiring fully 1∶1 or 10∶1 susceptible:resistant ratios in order to detect resistance. Our assay can also work in sputum so long as sufficient quantities of Tb are present (>1000 cfu/ml). This work demonstrates the utility of digital PCR to detect and quantify heteroresistance in drug resistant Tb, which may be useful to inform treatment decisions faster than agar proportion

Rapid First- and Second-Line Drug Susceptibility Assay for Mycobacterium tuberculosis Isolates by Use of Quantitative PCR▿

The slow turnaround time for Mycobacterium tuberculosis drug susceptibility results is a barrier to care. We developed a rapid quantitative PCR (qPCR)-based phenotypic antimicrobial susceptibility test that utilizes amplification of the M. tuberculosis 16S rRNA gene after 3 days of incubation with antituberculosis drugs. To decrease background from killed organisms, we used propidium monoazide (PMA), a DNA-binding dye that penetrates damaged bacterial cells and renders DNA unamplifiable. M. tuberculosis was cultured in broth media containing PMA with or without drugs for 3 days prior to DNA extraction and real-time PCR amplification. 16S rRNA qPCR exhibited a significant decrease in threshold cycle (CT) time values (CT control − CT drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as ΔCT or as 2ΔCT and with appropriate cutoffs yielded an accuracy of 89 to 100% on 38 susceptible, multidrug-resistant, and extensively drug-resistant strains compared with conventional agar proportion susceptibility results for isoniazid, rifampin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, linezolid, and cycloserine and compared with Bactec MGIT results for pyrazinamide. This PMA-qPCR assay is useful as a rapid 3-day first- and second-line drug susceptibility test for M. tuberculosis

Development of a TaqMan array card for pneumococcal serotyping on isolates and nasopharyngeal samples

Streptococcus pneumoniae is both a commensal and a major pathogen that causes invasive disease in people of all ages. The introduction of serotype-specific pneumococcal vaccines has reduced the burden of disease but has also led to replacement with new strains, thus serotyping remains important for vaccine related disease surveillance. Conventional serotyping methods are laborious and expensive. We developed an easy-to-perform genotypic TaqMan array card (TAC) to identify S. pneumoniae strains including lytA-based sequences and 53 sequence specific PCR reactions to identify 74 serotypes/serogroups covering all current vaccine types as well as prevalent non-vaccine types. The TAC method was evaluated on 146 clinical S. pneumoniae isolates and 13 non-pneumococcal species that naturally inhabit the upper respiratory tract, yielding 97% (142/146) sensitivity and 100% (13/13) specificity, versus standard Quellung serotyping. The calculated limit of detection was 20-200 fg (8-84 genome equivalents) per reaction. On 23 blinded nasopharyngeal specimens that were pneumococcus culture positive, the TAC pan-pneumococcus lytA assay was positive in 21 (91% sensitivity versus culture). On TAC lytA positive specimens, a serotype result was obtained 86% and was 95% accurate versus the subsequent culture’s Quellung result. TAC also detected mixed serotypes in 2 specimens where Quellung only detected the predominant serotype. This TAC method yields fast and comprehensive serotyping compared to the standard method and may be useful on direct specimens

Real-time PCR of undiluted DNA.

<p>The undiluted DNA of each H37Rv:XDR-TB mixture underwent real-time PCR for each gene with both probes. <i>katG</i> is shown, with detection of mutant sequence (blue trace) at H37Rv:XDR-TB mixtures of 0∶1, 1∶1, and late detection at 10∶1. Detection of wild-type sequence occurred at all H37Rv containing mixtures. qPCR thresholds were set per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone-0057238-g001" target="_blank">Figure 1</a> (red and blue horizontal lines for wild-type and mutant fluorophores, respectively). Similar results were found for other genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057238#pone.0057238.s001" target="_blank">Figure S1</a> for <i>rpoB</i>, <i>gyrA</i>, <i>rrs</i>).</p

Comparison of digital PCR, agar proportion, real-time PCR, and PCR with Sanger sequencing.

<p>R = resistant. S = susceptible. Agar proportion resistance defined as >1% resistant colonies, and the actual percentage of resistant colonies is shown in parentheses. Digital PCR resistance defined as >1% of PCR reactions indicating mutant sequence, with the actual percentage of PCR reactions with mutant sequence shown in parentheses. Real-time PCR resistance defined as mixed populations of wild-type and mutant sequence. Sequencing defined resistance as evidence of mutant sequence by chromatogram.</p