Quality of Sputum Specimen Samples Submitted for Culture and Drug Susceptibility Testing at the National Tuberculosis Reference Laboratory-Uganda, July-October 2013

Setting: The Uganda National Tuberculosis Reference Laboratory (NTRL) in Kampala. Objective: The proportion of poor quality specimens received for drug susceptibility testing (DST) at the NTRL and factors contributing to poor specimen quality were assessed. Design: A cross-sectional study was conducted of sputum samples received at the NTRL from patients at high risk for multi-drug-resistant tuberculosis (MDR TB) during July-October 2013. Demographic, clinical, and bacte-riological data were abstracted from laboratory records. A poor quality sample failed to meet any one of four criteria: ≥3 milliliter (ml) volume, delivered within 72 hours, triple packaged, and non-salivary appearance. Results: Overall, 365 (64%) of 556 samples were of poor quality;89 (16%) were not triple packaged, 44 (8%) were <3 mls, 164 (30%) were not delivered on time, and 215 (39%) were salivary in appearance. Poor quality specimens were more likely to be collected during the eighth month of TB treatment (OR = 2.5, CI = 1.2 - 5.1), from the East or Northeast zones (OR = 2.2, CI = 1.1 - 4.8), and from patients who previously defaulted from treatment (OR = 1.9, CI = 1.1 - 3.2). Conclusion: The majority of sputum samples had poor quality. Additional efforts are needed to improve quality of samples collected at the end of treatment, from East and Northeast zones, and from patients who had previously defaulted.

Inferring Mycobacterium Tuberculosis Drug Resistance and Transmission using Whole-genome Sequencing in a High TB-burden Setting in China

Objective China is among the 30 countries with a high burden of tuberculosis(TB)worldwide,and TB remains a public health concern.Kashgar Prefecture in the southern Xinjiang Autonomous Region is considered as one of the highest TB burden regions in China.However,molecular epidemiological studies of Kashgar are lacking.Methods A population-based retrospective study was conducted using whole-genome sequencing(WGS)to determine the characteristics of drug resistance and the transmission patterns.Results A total of 1,668 isolates collected in 2020 were classified into lineages 2(46.0%),3(27.5%),and 4(26.5%).The drug resistance rates revealed by WGS showed that the top three drugs in terms of the resistance rate were isoniazid(7.4%,124/1,668),streptomycin(6.0%,100/1,668),and rifampicin(3.3%,55/1,668).The rate of rifampicin resistance was 1.8%(23/1,290)in the new cases and 9.4%(32/340)in the previously treated cases.Known resistance mutations were detected more frequently in lineage 2 strains than in lineage 3 or 4 strains,respectively:18.6%vs.8.7 or 9%,P<0.001.The estimated proportion of recent transmissions was 25.9%(432/1,668).Multivariate logistic analyses indicated that sex,age,occupation,lineage,and drug resistance were the risk factors for recent transmission.Despite the low rate of drug resistance,drug-resistant strains had a higher risk of recent transmission than the susceptible strains(adjusted odds ratio,1.414;95%CI,1.023–1.954;P=0.036).Among all patients with drug-resistant tuberculosis(DR-TB),78.4%(171/218)were attributed to the transmission of DR-TB strains.Conclusion Our results suggest that drug-resistant strains are more transmissible than susceptible strains and that transmission is the major driving force of the current DR-TB epidemic in Kashgar.

Comparison of Two Molecular Assays For Detecting Smear Negative Pulmonary Tuberculosis

Objective To compare the performance of MTBDRplus V2 and Xpert MTB/RIF for detecting smear negative pulmonary tuberculosis （PTB）. Methods Clinical PTB suspects were enrolled consecutively in Anhui Chest Hospital and Xi＇an Chest Hospital from January to December in 2014. The sputum samples of smear negative PTB suspects were collected and decontaminated. The sediment was used to conduct MTBDRplus V2, Xpert MTB/RIF and drug susceptibility test （DST）. All the samples with discrepant drug susceptibility result between molecular methods and phenotypic method were confirmed by DNA sequencing. Results A total of 1973 cases were enrolled in this study. The detection rates of Mycobacterium tuberculosis complex （MTBC） by MTBDRplus V2 and Xpert MTB/RIF were 27.67% and 27.98%, respectively. When setting MGIT culture result as a gold standard, the sensitivity and specificity of MTBDRplus V2 were 86.74% and 93.84%, and the sensitivity and specificity of Xpert MTB/RIF were 86.55% and 93.43%, respectively. For the detection of the resistance to rifampin, the sensitivity and specificity of MTBDRplus V2 were 94.34% and 96.62%, and the sensitivity and specificity of Xpert MTB/RIF were 88.68% and 95.96%, respectively. For the detection of the resistance to isoniazid, the sensitivity and specificity of MTBDRplus V2 were 77.38% and 98.02%, respectively. Conclusion MTBDRplus V2 and Xpert MTB/RIF can be used to detect MTBC in smear negative samples with satisfactory performance.

Multicenter Evaluation of the Molecular Line Probe Assay for Multidrug Resistant Mycobacterium Tuberculosis Detection in China

In order to evaluate the performance of a molecular Hain line probe assay （Hain LPA） for rapid detection of rifampicin and isoniazid resistance of Mycobocterium tuberculosis in China, 1612 smear positive patients were consecutively enrolled in this study. Smear positive sputum specimens were collected for Hain LPA and conventional drug susceptibility testing （DST）. The sensitivity and specificity of Hain LPA were analyzed by using conventional DST as golden reference. The sensitivity, specificity, positive predictive value {PPV） and negative predictive value （NPV） for rifampicin resistance detection were 88.33%, 97.66%, 81.54%, and 98.62%, respectively. The sensitivity, specificity, PPV and NPV for isoniazid resistance detection were 80.25%, 98.07%, 87.25%, and 96.78%, respectively. These findings suggested that Hain LPA can be an effective method worthy of broader use in China.

Pre-Extensively Drug Resistant Tuberculosis (Pre-XDR-TB) among Pulmonary Multidrug Resistant Tuberculosis (MDR-TB) Patients in Bangladesh

Background & Objectives: Emergence of drug resistant Tuberculosis (TB) is a major obstacle in the TB control programme of Bangladesh. This study was carried out to detect pre-extensively drug resistant TB (pre-XDR-TB) cases among the multidrug resistant TB (MDR-TB) patients in Bangladesh, as the early detection of pre-XDR-TB can guide clinicians in the appropriate modification of MDR-TB treatment regimen with effective drugs to prevent treatment failure. Methodology: A total of 68 MDR-TB cases were enrolled in this study. Multiplex Real-time PCR was done to detect pre-XDR-TB cases directly from sputum samples of MDR-TB patients. Results: Out of 68 MDR-TB cases 11 (16.18%) cases were detected as pre-XDR-TB. The resistant profile of the 11 pre-XDR-TB revealed 9 (81.82%) cases of fluoroquinolone (FLQ) resistant pre-XDR-TB and 2 (18.18%) cases of injectable second line (ISL) agent resistant pre-XDR-TB. Out of 11 pre-XDR-TB cases 7 (63.64%) cases had history of taking treatment for MDR-TB regularly, 1 (9.09%) case had history of taking treatment for MDR-TB irregularly and 3 (27.27%) cases had no history of taking treatment for MDR-TB. Conclusion: This study encountered a high rate of pre-XDR-TB cases along with a significant number of primarily resistant bacilli which is of concern in the management of MDR-TB. It is evident that Bangladesh is in urgent need to device strategies for rapid and early detection of pre-XDR-TB in order to prevent treatment failure of MDR-TB cases and also to halt the progression of MDR-TB cases to extensively drug resistant TB (XDR-TB), which is not only difficult but also very expensive to treat.

Molecular characterization of the rpoB gene mutations of Mycobacterium tuberculosis isolated from China

Objective: To analyze characterization of the rpoBgene mutations of Mycobacterium tuber- culosis isolated from China and to explore the association of specific mutations conferring rifampicin (RIF) resistance with Beijing genotype strains. Methods: Genotypic analysis of 3479M. tuberculosis isolatesincluding 402 RIF-resistantand 3077 RIF-susceptible isolated from the na- tional drug-resistant tuberculosis baseline survey was performed. Results: DNA sequencing analysis of the 81-bp RIF resistance determining region (RRDR) of the ropB gene revealed that 98.01% of RIF-resistant strains showedrpoBgene mutation, isolates with mutations at codon rpoB531, rpoB 526 and rpoB 516 were the most frequently. Analysis of the rpoB gene of 3077 RIF-susceptible strains revealed that 98.96% of the strains had no mutation. The distribution of mutation frequency at differentcritical codons in different regions of China was statistically significant (p = 0.001). There was no significant difference in the occurrence of mutations at critical codons between the rifampicin-resistant Bei-jing and non-Beijing isolates.Conclusion: About 98% of RIF-resistant strains isolated from China carry mutations in RRDR ofrpoB gene.Mutation profiles in RIF-resistantM. tuberculosis clinical isolates are variable depending on the different geographical regionsof China. The results provide valuable information in adopting new molecular methods for diagnosis of TB in China.

First-Line Drug Resistance Patterns of Mycobacterium tuberculosis Complex Isolates from Re-Treatment Patients from Sudan

Drug susceptibility testing (DST) plays a pivotal role in TB patients’ management leading to the selection of most effective drugs. This study aimed to determine resistance patterns to first line anti-TB drugs in Mycobacterium tuberculosis isolates from re-treated patients from Sudan. A total of 239 sputum specimens were collected from smear positive re-treatment TB patients during the period from July 2009 to July 2010. Specimens were pre-treated according to Petroff method. The recovered isolates were tested for sensitivity to first line anti-TB drugs by the 1% proportion method. One hundred and forty three (143/239, 59.8%) mycobacterial isolates were successfully recovered. The majority (98.6%, 141/143) of the isolates were Mycobacterium tuberculosis complex strains. Two strains (2/143, 1.4%) were identified as RIF/INH-resistant MOTT, while fifty four isolates (38.3%, 54/141) were MDR. Multi- drug resistant Mycobacterium tuberculosis complex (MDR-TB) among re-treatment patients from national referral centers for tuberculosis diagnosis and management was considerably high in the study isolates.

The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology

Avian pathogenic Escherichia coli(APEC)belonging to extraintestinal pathogenic E.coli(ExPEC)can cause severe infections in extraintestinal tissues in birds and humans,such as the lungs and blood.MprA(microcin production regulation,locus A,herein renamed AbsR,a blood survival regulator),a member of the MarR(multiple antibiotic resistance regulator)transcriptional regulator family,governs the expression of capsule biosynthetic genes in human ExPEC and represents a promising druggable target for antimicrobials.However,a deep understanding of the AbsR regulatory mechanism as well as its regulon is lacking.In this study,we present a systems-level analysis of the APEC AbsR regulon using ChIP-Seq(chromatin immunoprecipitation sequencing)and RNA-Seq(RNA sequencing)methods.We found that AbsR directly regulates 99 genes and indirectly regulates 667 genes.Furthermore,we showed that:1)AbsR contributes to antiphagocytotic effects by macrophages and virulence in a mouse model for systemic infection by directly activating the capsular gene cluster;2)AbsR positively impacts biofilm formation via direct regulation of the T2SS(type II secretion system)but plays a marginal role in virulence;and 3)AbsR directly upregulates the acid tolerance signaling system EvgAS to withstand acid stress but is dispensable in ExPEC virulence.Finally,our data indicate that the role of AbsR in virulence gene regulation is relatively conserved in ExPEC strains.Altogether,this study provides a comprehensive analysis of the AbsR regulon and regulatory mechanism,and our data suggest that AbsR likely influences virulence primarily through the control of capsule production.Interestingly,we found that AbsR severely represses the expression of the type I-F CRISPR(clustered regularly interspaced short palindromic repeats)-Cas(CRISPR associated)systems,which could have implications in CRISPR biology and application.

Comparing the Genotype and Drug Susceptibilities between Mycobacterium avium and Mycobacterium intracellulare in China

Objective Mycobacterium avium （M. avium） and Mycobacterium intracellulare （M. intracellulare） are the major causative agents of nontuberculous mycobacteria （NTM）-related pulmonary infections. However, little is known about the differences in drug susceptibility profiles between these two species. Methods A total of 393 NTM isolates were collected from Shanghai Pulmonary Disease Hospital. Sequencing of partial genes was performed to identify the strains at species level. The minimum inhibitory concentration （MIC） was used to evaluate the drug susceptibility against 20 antimicrobial agents. Variable number of tandem repeat （VNTR） typing was conducted to genotype these two species. Results A total of 173 （44.0%） M. avium complex （MAC） isolates were identified, including 41 （10.4%） M. avium isolates and 132 （33.6%） M. intracellulare isolates. Clarithromycin and amikacin were the two most effective agents against MAC isolates. The Hunter-Gaston Discriminatory Index （HGDI） values for VNTR typing of M. avium and M. intracellulare isolates were 0.993 and 0.995, respectively. Levofloxacin resistance was more common among the unclustered strains than among the clustered strains of M. intracellulare. Conclusion M. intrecellulare was the most common NTM species in China. Clarithromycin and amikacin had high antimicrobial activities against MAC. VNTR typing of MAC isolates revealed a high discriminatory power. Levofloxacin resistance was associated with unclustered strains of M. intracellulare.

Antimicrobial Susceptibility Testing and Molecular Characterization of Mycobacterium fortuitum Isolates in China

We performed molecular identification of clinical isolates of Mycobacterium fortuitum（M. fortuitum） and conducted drug susceptibility testing to analyze the in vitro susceptibility of clinical M. fortuitum isolates and potential molecular mechanism conferring resistance to fluoroquinolone and macrolide drugs. The results showed that moxifloxacin had the highest in vitro activity against M. fortuitum, and most M. fortuitum isolates were resistant to clarithromycin and linezolid in China. The loss of genetic mutation in clarithromycin-and amikacin-resistant isolates indicates that some other intrinsic mechanism conferring clarithromycin and amikacin resistance plays an essential role in M. fortuitum infection.

Evaluation of the Third 90 of the 90-90-90 Cascade for the Period 2019-2020 in the Central African Republic

Introduction In Central-African Republic, according to UNAIDS in 2019, out of approximately 100,000 people living with HIV, 70% (72,000) knew their HIV status and 47,000 (46%) were on ARV therapy;however, there is a paucity of data on viral load suppression in people on ARV therapy. The objective of this study was to assess the third 90 of the UNAIDS strategy for the years 2019 and 2020 in the CAR. Methods We analyzed the available viral load data extracted from the data base of the medical analysis laboratory (SYSLAM) of the Institut Pasteur of Bangui for the years 2019 and 2020. The viral loads were determined based on plasma collected in an EDTA tube with Cepheid’s GeneXpert® 16-module controllers. Viral load data were extracted from SYSLAM, converted to Excel format, and analyzed with STATA version 14 software. The significance threshold for the statistical tests was set at 5%. Results This study included 22,895 patients, of who 72% were female. The average age was 40.82 years, and the majority of the patients (80%) came from the city of Bangui. Regarding the virological parameters associated with this study, 66% of the patients had significant viral load suppression according to the WHO recommendations and 34% were in virological failure. Patients over 50 years of age (71.85%) and age group 40 - 49 years (69.25%) recorded significant levels of viral load suppression. On the other hand, 63.45% of patients under 18 years of age had virological failure. All of these results were statistically significant (p < 0.005). Conclusion There should be a concerted effort, to make viral load accessible and available to all patients receiving ARV treatment in the CAR and the management of HIV/AIDS infection of children and adolescents should be given special attention.

Rating of CCR5-Delta 32 Homozygous Mutation in Sudanese HIV Patients and Sex Workers

Background: Prevention against human immunodeficiency virus (HIV) includes natural resistance in the population;mainly frequency of cysteine-cysteine chemokine receptor type-5 (CCR5-delta 32 mutation). By knowing the frequency of this resistance in the community, the proportion of the population susceptible to infection can be determined. This study aimed to detect for the first time the rate of CCR5-delta 32 mutation in Sudanese individuals with HIV and sex workers. Methods: Cross-sectional study was followed in the parade from 2019 through 2021, study groups were Sudanese with HIV and sex workers. Sero-negativity of sex workers was confirmed by a rapid immunochromatography test (ICT). A blood sample was targeted for DNA isolation. PCR amplification was accomplished for CCR5 wild type and CCR5-delta 32 mutation genes using specific primers. Result: Among HIV patients, males, basic education level and ages below 60 years were commonly recorded while ages below 40 years, secondary education level and single marital status were predominated in sex workers. All HIV patients were positive for CCR5 wild type and negative for CCR5-delta 32 genotype. The sex workers group showed a frequency of 3.5% (97/200) for homozygous CCR5-delta 32 mutation. Conclusion: The rating of homozygous CCR5-delta 32 genotype in studied Sudanese sex workers was relatively more than other results obtained from African countries, and the mutation was significantly detected among sex workers group (P value = 0.008) when compared to the studied HIV group.

Characterization of isoniazid resistance and genetic mutations in isoniazid-resistant and rifampicin-susceptible Mycobacterium tuberculosis in China

Background Patients with tuberculosis resistant to isoniazid but susceptible to rifampicin(Hr-Rs TB)remain a neglected demographic,despite a high disease burden and poor outcomes of these patients.The aim of this study was to investigate the characteristics of isoniazid-resistance-related mutations in Mycobacterium tuberculosis and resistance rates to drugs included in WHO-recommended regimens for Hr-Rs patients.Methods Mycobacterium tuberculosis isolates(n=4922)obtained from national tuberculosis drug-resistance surveillance were subjected to whole-genome sequencing to identify Hr-Rs strains.The minimal inhibitory concentrations(MICs)were established for the Hr-Rs strains to determine the isoniazid resistance levels.We also identified drug-resistance-associated mutations for five drugs(fluoroquinolones,ethambutol,pyrazinamide,streptomycin,and amikacin)in the Hr-Rs strains.Results Of the 4922 strains,384(7.8%)were Hr-Rs.The subculture of seven strains failed,so 377(98.2%)strains underwent phenotypic MIC testing.Among the 384 genotypic Hr-Rs strains,242(63.0%)contained the katG Ser315Thr substitution;115(29.9%)contained the-15C>T in the promoter region of the fabG1 gene;and 16(4.2%)contained Ser315Asn in the katG gene.Of the 239 strains with the Ser315Thr substitution,229(95.8%)had MIC≥2µg/mL,and of the 114 strains with the-15C>T mutation,103(90.4%)had 0.25µg/mL≤MIC≤1µg/mL.The genotypic resistance rates were 0.8%(3/384)for pyrazinamide,2.3%(9/384)for ethambutol and fluoroquinolones;39.6%(152/384)of the strains were resistant to streptomycin,but only 0.5%(2/384)of the strains were resistant to amikacin.Conclusion Ser315Thr in katG was the predominant mutation conferring the Hr-Rs phenotype,followed by the fabG1-15C>T mutation.The combination of rifampicin,pyrazinamide,ethambutol,and levofloxacin should be effective in the treatment of patients with Hr-Rs tuberculosis because the resistance rates for these drugs in China are low.

<i>gyrA</i>Gene Mutation Conferring Phenotypic Cross-Resistance among Fluoroquinolones (Ofloxacin, Levofloxacin and Gatifloxacin) in Multidrug Resistant <i>Mycobacterium tuberculosis</i>Strains Isolated from Pulmonary MDR-TB Patients in Bangladesh

Background and objectives: Fluoroquinolones (FLQs) are an essential component of multidrug resistant-tuberculosis (MDR-TB) treatment regimen but unfortunately the emergence of FLQ resistant MDR-TB cases is challenging the current MDR-TB treatment regimen. FLQ resistance is mainly caused by gyrA gene mutation and phenotypic cross-resistance may occur among the different FLQs. A limited number of data exists regarding the cross-resistance phenomenon among FLQs and it appears that resistance to the present class representative FLQ, ofloxacin (OFX), may or may not correlate with complete cross-resistance to other FLQs. So the study was designed to observe if gyrA gene mutations confer to the phenotypic cross-resistance among FLQs [OFX, Levofloxacin (LFX) and Gatifloxacin (GFX)] tested. Methodology: Sputum samples from 68 diagnosed pulmonary MDR-TB cases were collected. All samples were subjected to Multiplex Real-time PCR for the detection of gyrA gene mutations and conventional culture on Lowenstein-Jensen (L-J) media followed by drug sensitivity testing (DST) of culture isolates (MDR-TB strains) by indirect proportion method for the detection of phenotypic resistance pattern to OFX, LFX and GFX. Results: Out of the 68 MDR-TB sputum samples 13 (19.11%) had MDR-TB bacilli with mutations in the gyrA gene and 11(16.18%) of the MDR-TB culture isolates were found to have resistance to FLQs by DST. The study observed that 11 MDR-TB samples with gyrA gene mutations showed complete phenotypic cross-resistance among OFX, LFX and GFX. Conclusion: This study found that mutation in the gyrA gene of the MDR-TB bacilli results in the complete cross-resistance among the FLQs (OFX, LFX and GFX) tested. It is therefore of utmost importance to carry out the base line resistance and cross-resistance tests for the individual FLQs prior to initiating the treatment of MDR-TB cases in Bangladesh for successful clinical outcomes.

Evaluation of the Xpert MTB/RIF assay for diagnosis of tuberculosis and rifampin resistance in county-level laboratories in Hunan province, China

Background The Xpert MTB/RIF showed high sensitivity and specificity in previous studies carried out in different epidemiological and geographical settings and patient populations in high-burden tuberculosis （TB） countries.However,there were little data obtained by validation or demonstration study of the assay in China.In this study,the performance of Xpert MTB/RIF was investigated in two county-level laboratories in Hunan Province,China.Methods Consecutive patients with suspected pulmonary tuberculosis （PTB） and suspicion for multidrug-resistant tuberculosis （MDR-TB） were enrolled.For each patient suspected to have PTB,three sputum specimens （one spot sputum,one night sputum,and one morning sputum） were collected and each sputum was tested with smear microscopy,L（o）wenstein-Jensen （LJ） culture,and Xpert MTB/RIF test.For comparison across subgroups and testing methods,95％ confidence intervals were calculated.All analyses were done with SPSS 16.0,and P ＜0.05 was regarded as significant.Results For case detection,the sensitivity of Xpert MTB/RIF was 100％ for smear-and culture-positive TB and 88.6％ for smear-negative and culture-positive TB; the overall sensitivity was 94.5％ for all culture-positive patients.The specificity was 99.8％.The sensitivity of Xpert MTB/RIF assay was 22.0％ in clinical TB patients and the specificity reached 100.0％ in the group of patients who are infected with nontuberculous mycobacteria.For the detection of rifampin resistance,the sensitivity of MTB/RIF RIF-resistance detection was 92.9％,and the specificity was 98.7％.Of the 26 Xpert MTB/RIF-positive and RIF-resistant patients confirmed by LJ proportion tests,20 （76.9％） patients were infected by MDR-TB.Conclusions The Xpert MTB/RIF assay is a highly sensitive and specific method for diagnosis of TB and RIF resistance,which will enable it to have the potential to be used in county-level laboratories and lead to the reduction of the infectious pool and improvements in TB control in China.Further evaluations in county-level laboratories for implementing the assay are still required.

Characteristics of compensatory mutations in the rpoC gene and their association with compensated transmission of Mycobacterium tuberculosis

The aim of this study was to characterize rpoC gene mutations in Mycobacterium tuberculosis(MTB)and investigate the factors associated with rpoC mutations and the relation between rpoC mutations and tuberculosis(TB)transmission.A total of 245 MTB clinical isolates from patients with TB in six provinces and two municipalities in China were characterized based on gene mutations through DNA sequencing of rpoC and rpoB genes,phenotyping via standard drug susceptibility testing,and genotypic profiling by mycobacterial interspersed repetitive unit-variable number tandem repeat(MIRU-VNTR)typing.Approximately 36.4%of the rifampinresistant isolates harbored nonsynonymous mutations in the rpoC gene.Twenty-nine nonsynonymous single mutations and three double mutations were identified.The rpoC mutations at locus 483(11.3%)were predominant,and the mutations at V483G,W484G,I491V,L516P,L566R,N698K,and A788E accounted for 54.5%of the total detected mutations.Fifteen new mutations in the rpoC gene were identified.Rifampin resistance and rpoB mutations at locus 531 were significantly associated with rpoC mutations.MIRU-VNTR genotype results indicated that 18.4%of the studied isolates were clustered,and the rpoC mutations were not significantly associated with MIRU-VNTR clusters.A large proportion of rpoC mutation was observed in the rifampicin-resistant MTB isolates.However,the findings of this study do not support the association of rpoC mutation with compensated transmissibility.