Drug Resistance Surveillance Research Articles

Antibiotic Susceptibility Patterns of Bacteria Isolated from Hospital Surfaces and Environment in Kenya

Objective: Control of hospital environment is key to success of healthcare quality. Increasing emergence and spread of pathogenic bacteria is of great concern and continues to challenge infection prevention and epidemiology practice. This study aimed at providing information about the management of hospital environment and wastes in selected hospitals in Kenya, determine prevalence of pathogenic bacteria and their antibiotic susceptibility.
 Methods: A cross sectional study was conducted at Kenyatta National Hospital (KNH) (public) and Kikuyu Mission Hospital (KMH) (private) in Kenya from May 2015 to April 2017. In microbiological analysis, a total of 246 samples from each of the two hospitals was obtained using sterile cotton swabs from random sampling of hospital different surfaces, drainages, hands of healthcare givers and hospital waste dump site among others.
 Results: A total of 471 bacterial isolates were recovered, and were distributed as follows; Providentia spp, Staphylococcus aureus spp, Escherichia coli spp (E. coli), other Gram negative bacteria were, Pseudomonas spp, coagulase negative Staphylococcus (CONS), Serratia spp, Klebsiella spp, Proteus spp and Enterobacter spp. Susceptibility test revealed that Escherichia coli isolates were the most sensitive isolate to antibiotics. Imipenem drug showed 100% sensitivity for Gram negative, while Gram-positive isolates, linezolid antibiotic was the most sensitive drug.
 Discussion: There is need for stringent review of hospital waste management system in Kenya. The frequency of ESBL producing strains among clinical isolates has been steadily increasing.
 Conclusion: Continued drug resistance surveillance of ESBL isolates is necessary to guide the appropriate and judicious antibiotic use.

Detection of H275Y Mutation Conferring Oseltamivir Drug Resistance in Influenza A (H1N1) pdm09 Virus

In the treatment of influenza, Neuraminidase inhibitors (NAIs) (Oseltamivir and Zanamivir) play a major role. The emergence of variants of influenza A (H1N1) pdm09 virus resistant to Oseltamivir is a matter of great concern as it limits its usage. Therefore, vigilant monitoring for Oseltamivir-resistant viruses has been recommended by the World Health Organization (WHO). Our study aimed to screen the influenza A (H1N1) pdm09 virus for NAI drug resistance during the outbreak of 2015-16 in North-Western India. A total of 640 H1N1pdm09 virus-positive samples were screened for drug resistance to Oseltamivir by WHO allelic discrimination real-time RT-PCR protocol. The allelic discrimination PCR protocol can detect the presence of single nucleotide polymorphisms (SNPs), the H275Y mutation is detected by this method which causes resistance to Oseltamivir. Sanger sequencing of partial fragment of NA gene (fragment IV), of 90 samples were performed to confirm the presence of NA-H275Y mutation. Neuraminidase susceptibility of 20 randomly selected isolates to Oseltamivir was tested using NA inhibition chemiluminiscence based assay. Among 640 H1N1pdm09 positive samples tested, H275Y mutation was detected in one sample (0.15%) by PCR and confirmed by Sanger sequencing also. All the 20 isolates tested for NAI susceptibility by NA star assay were found to be sensitive to Oseltamivir. WHO allelic discrimination PCR is an easy, rapid and sensitive method for high-throughput detection of resistance to Oseltamivir. Systematic regular drug resistance surveillance of Influenza A is essential to monitor the emergence and spread of drug-resistant strains.

Screening for antifolate and artemisinin resistance in Plasmodium falciparum clinical isolates from three hospitals of Eritrea

Background: Antimalarial drug resistance is a major challenge hampering malaria control and elimination. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies. This study sought to verify the genetic mechanism of resistance to sulfadoxine-pyrimethamine and assess the occurrence of point mutations associated with artemisinin resistance in P. falciparum clinical isolates from Eritrea. Methods: Nineteen dried blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, Eritrea. The patients were followed up after receiving treatment with first line artesunate-amodiaquine. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the P. falciparum bifunctional dihydrofolate reductase-thymidylate synthase (Pfdhfr, PF3D7_0417200), dihydropteorate synthase (Pfdhps, PF3D7_0810800) and kelch 13 (PfK13, PF3D7_1343700) genes. Results: Eight of nineteen (42%) of the dried blood spot samples were successful for PCR-amplification. Data analyses of the PCR-positive isolates revealed the following point mutations: Pfdhfr N51I in four isolates, C59R in one isolate, S108N in four isolates, a rare non-synonymous substitution V45A in four isolates and Pfdhps K540E in four isolates. No PfK13 point mutations were reported. Conclusions: Pfdhfr C59R and Pfdhps K540E point mutations are reliable markers for the sulfadoxine-pyrimethamine quintuple mutant haplotype combination. These findings highlight first reports in Eritrea, which verify the underlying genetic mechanism of antifolate resistance. Continuous monitoring of the PfK13 marker is recommended.

Monitoring Emerging Human Immunodeficiency Virus Drug Resistance in Sub-Saharan Africa in the Era of Dolutegravir

Dolutegravir-based regimens are now standard of care for human immunodeficiency virus treatment for millions of people around sub-Saharan Africa. To ensure its continued efficacy, monitoring of emerging drug resistance that inform a treatment strategy among those failing is crucial. In this report, we outline the US President's Emergency Plan for AIDS Relief to leverage viral load infrastructure to implement effective drug resistance surveillance in the countries it supports.

Plasmodium falciparum kelch 13 Mutations, 9 Countries in Africa, 2014-2018.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014–2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Characteristics of Four Novel Recombinant Strains from the Backbone of CRF55_01B and CRF65_cpx in Beijing by Near Full-Length Genome.

The HIV-1 epidemic was mainly driven by men who have sex with men (MSM) recently in Beijing, China, with high genetic diversity. Novel recombinant strains were frequently reported at 3.4%-9.9%. It is imperative to interpret the recombinant modes and the putative transmission sources by near full-length genome (NFLG). Four individuals from the MSM population were identified as novel recombinant strains during surveillance of pretreatment drug resistance. NFLG sequences were harvested by near end-point dilution and nested PCR with two overlapping half fragments. Phylogenetic inference was performed with subtyping reference sequences and major parental strain sequences, to explore the patterns of genetic recombinant and potential sources of parent strains. The breakpoints were determined using SimPlot 3.5 to draw genome mosaic map, and the potential parental strains were confirmed by Mega 6.0 using segmental neighbor-joining trees. BL19487-00 and BL1948-00 sequences were obtained from epidemiologically linked individuals and shared similar breakpoints (HXB2 nt 4,497 ± 8 to 4,722) with substitution of subtype B pol gene segment in the backbone of CRF55_01B. BL3104-00 and BL4307-00 carried seven and eight breakpoints, respectively, in the backbone of CRF65_cpx with g5 CRF01_AE substitutions. The recombinant fragments were located around gag, pol, and env genes, with vpr-tat and nef-3'-LTR genes only for BL4307-00. No transmitted drug resistance was observed with the four unique recombinant forms (URFs), except for some drug resistance associated mutations. The advent of URFs around CRF55_01B and CRF65_cpx identified in recent years implied that the sexual behaviors were active and the epidemic of HIV was complicated among MSM in Beijing. Molecular epidemiological surveillance and precise control should be reinforced for this population.

CBNAAT Based Identification and Demographic Pattern of Drug Resistant Mycobacterium Tuberculosis in South Kashmir, India- a One Year Retrospective Study

India is the highest TB burden country in the world having an estimated incidence of 26.9 lakh cases in 2019. With a population of 1.32 billion, India has the highest burden of drug resistant TB (DR-TB) in the world. North zone of India is the second highest MDR-TB prevalent zone after the West zone of India. MDR TB treatment involves prolonged treatment with injectable second-line drugs, associated with more adverse effects, suboptimal treatment outcomes and higher risks of mortality compared to patients with drug-sensitive TB and those with lesser resistant forms of TB.
 Materials methods: This retrospective study was conducted in the department of Microbiology Government Medical College Anantnag, data was analyzed from March 2017 to February 2018. Non-sterile specimens were processed by Modified Petroff Method. Sterile specimens were concentrated by centrifugation and smear and cultures was inoculated from the sediment. CBNAAT assay was performed by Gene Xpert (Cepheid) 4 system according to the manufacturers’ recommendations.
 Results: Of the total 1497 clinically suspected tuberculosis specimens collected, 1370 (91.5%) were pulmonary and 127 (8.5%) were presumptive extra pulmonary tuberculosis received from different anatomical sites. Maximum clustering of cases was seen in 10-20 years age group. Out of the total 1497 samples 200 were CBNAAT confirmed Mycobacterium Tuberculosis positive samples. In which 155 were pulmonary and 45 were extra pulmonary. The average percentage positivity rate (i.e. percentage of MTB positive samples out of total samples tested) was 13.3% (200/1497). Rifampicin resistance (RR-TB) was seen in 5.5% (11/200) samples. Out of the samples detected positive (200): 155 were pulmonary samples and out of these 155 pulmonary samples 8 were found to be RR MTB 5.1% (8/155). Also out of the 200 positive samples 45 were extra pulmonary and out of these 45 extra pulmonary samples 3 were found to be RR MTB 6.6% (3/45).
 Conclusion: In this study we found that in our region 5.5% cases of TB were RR-TB, 3.2% were new cases and 13% RR-TB was seen in previously treated cases of MTB. The screening of drug resistance has to be expanded to offer universal DST including expanded DST .The second and most important activity is to strengthen drug resistance surveillance under the various national programs with inclusion of laboratories in the private sector as well. The state level regional studies also give us the opportunity to plan and execute intervention prioritization, based on the drug resistance trends observed.

Plasmodium falciparum resistance to ACTs: Emergence, mechanisms, and outlook

Emergence and spread of resistance in Plasmodium falciparum to the frontline treatment artemisinin-based combination therapies (ACTs) in the epicenter of multidrug resistance of Southeast Asia threaten global malaria control and elimination. Artemisinin (ART) resistance (or tolerance) is defined clinically as delayed parasite clearance after treatment with an ART drug. The resistance phenotype is restricted to the early ring stage and can be measured in vitro using a ring-stage survival assay. ART resistance is associated with mutations in the propeller domain of the Kelch family protein K13. As a pro-drug, ART is activated primarily by heme, which is mainly derived from hemoglobin digestion in the food vacuole. Activated ARTs can react promiscuously with a wide range of cellular targets, disrupting cellular protein homeostasis. Consistent with this mode of action for ARTs, the molecular mechanisms of K13-mediated ART resistance involve reduced hemoglobin uptake/digestion and increased cellular stress response. Mutations in other genes such as AP-2μ (adaptor protein-2 μ subunit), UBP-1 (ubiquitin-binding protein-1), and Falcipain 2a that interfere with hemoglobin uptake and digestion also increase resistance to ARTs. ART resistance has facilitated the development of resistance to the partner drugs, resulting in rapidly declining ACT efficacies. The molecular markers for resistance to the partner drugs are mostly associated with point mutations in the two food vacuole membrane transporters PfCRT and PfMDR1, and amplification of pfmdr1 and the two aspartic protease genes plasmepsin 2 and 3. It has been observed that mutations in these genes can have opposing effects on sensitivities to different partner drugs, which serve as the principle for designing triple ACTs and drug rotation. Although clinical ACT resistance is restricted to Southeast Asia, surveillance for drug resistance using in vivo clinical efficacy, in vitro assays, and molecular approaches is required to prevent or slow down the spread of resistant parasites.

Pretreatment HIV Drug-resistance Surveillance as a Tool for Monitoring and Control of the HIV/AIDS Epidemic in Cuba.

The HIV/AIDS epidemic is an ongoing threat to public health. Its elimination requires greater efforts to broaden antiretroviral treatment coverage, availability and personalization. HIV drug resistance is currently a global problem due to its continuing increase in recent years, undermining efficacy of antiretroviral therapy. Pretreatment HIV drug-resistance surveillance is part of WHO's strategy for addressing antiretroviral drug resistance. This paper describes and analyzes pretreatment HIV drug-resistance surveillance in Cuba. It presents a chronology of HIV resistance studies in untreated patients, along with their results and programmatic actions related to first- and second-line treatment regimens. Cuba's incorporation into the Global HIV Drug Resistance Surveillance Laboratories Network and the advantages of having a WHO-designated laboratory in which to conduct periodic studies of HIV drug-resistance surveillance are described. HIV drug-resistance surveillance in Cuba is a necessary tool in HIV/AIDS monitoring and control, as it obtains population-scale data used to inform programmatic decisions related to optimizing first- and second-line treatments for children and adults, as well as helping meet goals of eliminating HIV transmission.

Whole Genome Sequencing for Surveillance of Tuberculosis Drug Resistance in China: Based on a Cross-Sectional Surveillance Study

Background: Phenotypic drug susceptibility testing for prediction of tuberculosis (TB) drug resistance is slow and unreliable, limiting individualized therapy and monitoring of national TB data. Our study evaluated whole genome sequencing (WGS) for its predictive accuracy, use in TB drug-resistance surveillance, and ability to quantify the effects of resistance-associated mutations on minimum inhibitory concentrations (MICs) of anti-TB drugs. Methods: We used WGS to measure the susceptibility of 4880 isolates to ten anti-TB drugs ; for pyrazinamide, we used BACTEC MGIT 960. We determined the accuracy of WGS by comparing the prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. We used the Student-Newman-Keuls test to confirm MIC differences of mutations. Findings: Resistance to isoniazid, rifampin, and ethambutol were correctly predicted with at least 92·29% sensitivity, resistance to pyrazinamide with 50·52% sensitivity, and resistance to six second-line drugs with 85·05% to 96·01% sensitivity. In addition to the large overlap in estimated drug resistance prevalence by WGS and phenotypic testing, WGS can detect low-level resistant or sub-ECOFF mutations which may be missed by phenotyping. For nearly all drugs, resistance-conferring mutations had varying levels of impact on MICs. Interpretation: WGS can predict phenotypic susceptibility with high accuracy and allow for detection of new escape mutations by rapid molecular tools; as such, it can be a valuable tool in TB drug resistance surveillance and for determining the therapeutic approach. Funding Information: We acknowledge the financial supports of the grants from the Ministry of Science and Technology, the National Science and Technology Major Project (2018ZX10103001) and the National Key Research and Development Plan (No. 2020YFA0907200, 2019YFC0840602). Declaration of Interests: All authors report no conflicts of interest relevant to this article.

HIV-1 drug resistance and subtypes in newly reported HIV/AIDS patients before antiretroviral therapy in Taizhou city, 2016-2018

Objective: To explore the HIV-1 drug resistance and subtypes in newly reported HIV/AIDS patients before antiretroviral therapy (ART) in Taizhou city. Methods: A cross-sectional study of HIV-1 drug resistance was conducted among newly reported HIV/AIDS patients before ART in Taizhou from January 2016 to December 2018. HIV-1 pol gene sequences were obtained by RT-PCR. The sequences were submitted to the Stanford University drug resistance database. The drug resistance mutation and the sensitivity of HIV-1 strains to antiretroviral drugs were determined according to WHO Guidelines on HIV drug resistance surveillance of 2014. Results: A total of 806 HIV-1 pol gene sequences were obtained successfully. The overall HIV-1 drug resistance rate was 2.9% (23/806), 1.9% for non-nucleoside reverse transcriptase inhibitors (NNRTIs), 0.6% for nucleoside reverse transcriptase inhibitors (NRTIs), and 0.0% for protease inhibitors (PIs), respectively. From 2016 to 2018, the HIV-1 drug resistance rate was 1.6%, 1.8%, and 4.8%, respectively. The resistance mutations of NNRTIs and NRTIs were mainly K103 N (0.7%) and M184I/V (0.5%). HIV-1 subtypes were mostly CRF01_AE (42.7%,344/806),CRF07_BC (28.9%,233/806) and CRF08_BC (11.2%,90/806).HIV-1 subtypes among homosexually transmitted infections were mostly CRF01_AE (53.3%, 136/255) and CRF07_BC (32.2%, 82/255), and HIV-1 subtypes among heterosexually transmitted infections were mainly CRF01_AE (37.7%, 203/539), CRF07_BC (27.5%, 148/539) and CRF08_BC (16.1%, 87/539). Conclusion: HIV-1 drug resistance rate among newly reported HIV/AIDS patients before ART remained low in Taizhou during 2016 to 2018, an increasing trend seemed to be notifiable and warrants continuous surveillance of HIV-1 drug resistance.

Undisclosed exposure to antiretrovirals prior to treatment initiation: An exploratory analysis.

BackgroundThe proportion of individuals with a history of exposure (‘pre-exposure’) to antiretrovirals (ARVs) prior to formal initiation into antiretroviral treatment (ART) is unknown.ObjectivesThis study describes the detection of ARVs in plasma and/or hair, of persons who self-reported no pre-exposure to ART at their first-time initiation onto ART in three clinics in the province of Limpopo, South Africa (SA).MethodConcentrations of tenofovir (TDF), emtricitabine (FTC) and efavirenz (EFV) in the plasma and hair of individuals initiating ART were analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Next generation sequences of HIV polymerase gene were analysed with Geneious software 11.15, and drug resistance (DR) mutations were determined according to the Stanford HIV Drug-Resistance database. Participants’ demographic data were collected on a structured questionnaire. Data that describe prior exposure to ARV were also collected by this self-reporting method.ResultsPaired blood and hair samples were collected from 77 individuals newly initiated onto ART from 2017 to 2019. We detected at least one of the drugs in the plasma or hair of 41/77 (53.2%) patients who responded with a ‘no’ to the question ‘have you received ARVs before initiation onto ART?’ Thirty-one participants (n = 31/77, 40.3%) had TDF in either plasma or hair. Emtricitabine and EFV were found in the plasma or hair of 12/77 (15.6%) and 25/77 (32.4%) of participants respectively. Six (n = 6/77, 7.792%) had all three ARVs in plasma or hair. Prevalence of DR mutations at the > 5% significance threshold level in those known to have had ARV-exposure determined by LC-MS/MS prior to ART-initiation was not significant (χ2 = 0.798; p = 0.372), when compared to those who had no prior exposure but still showed DR.ConclusionAntiretroviral levels in the hair of individuals initiating treatment imply prolonged prior-exposure to that ARV. The presence of ARV in plasma and hair of persons living with HIV (PLWH) who deny ARV-use, requires an explanation. A larger study at multiple sites and regular DR surveillance of ART-naïve PLWH will be necessary to confirm the generalisability of these findings to the wider South African population.

Molecular surveillance of drug resistance: Plasmodium falciparum artemisinin resistance single nucleotide polymorphisms in Kelch protein propeller (K13) domain from Southern Pakistan

BackgroundK13 propeller (k13) polymorphism are useful molecular markers for tracking the emergence and spread of artemisinin resistance in Plasmodium falciparum. Polymorphisms are reported from Cambodia with rapid invasion of the population and almost near fixation in south East Asia. The study describes single nucleotide polymorphisms in Kelch protein propeller domain of P. falciparum associated with artemisinin resistance from Southern Pakistan.MethodsTwo hundred and forty-nine samples were collected from patients with microscopy confirmed P. falciparum malaria attending Aga Khan University Hospital during September 2015-April 2018. DNA was isolated using the whole blood protocol for the QIAmp DNA Blood Kit. The k13 propeller gene (k13) was amplified using nested PCR. Double-strand sequencing of PCR products was performed using Sanger sequencing methodology. Sequences were analysed with MEGA 6 and Bio edit software to identify specific SNP combinations.ResultsAll isolates analysed for k13 propeller allele were observed as wild-type in samples collected post implementation of ACT in Pakistan. C580Y, A675V, Y493H and R539T variants associated with reduced susceptibility to artemisinin-based combination therapy (ACT) were not found. Low frequency of M476I and C469Y polymorphisms was found, which is significantly associated with artemisinin resistance.ConclusionLow frequencies of both nonsynonymous and synonymous polymorphisms were observed in P. falciparum isolates circulating in Southern Pakistan. The absence of known molecular markers of artemisinin resistance in this region is favourable for anti-malarial efficacy of ACT. Surveillance of anti-malarial drug resistance to detect its emergence and spread need to be strengthened in Pakistan.

Impact of pre-existing drug resistance on risk of virological failure in South Africa.

There is conflicting evidence on the impact of pre-existing HIV drug resistance mutations (DRMs) in patients infected with non-B subtype virus. We performed a case-cohort substudy of the AIDS Drug Resistance Surveillance Study, which enrolled South African patients initiating first-line efavirenz/emtricitabine/tenofovir. Pre-ART DRMs were detected by Illumina sequencing of HIV pol and DRMs present at <20% of the viral population were labelled as minority variants (MVs). Weighted Cox proportional hazards models estimated the association between pre-ART DRMs and risk of virological failure (VF), defined as confirmed HIV-1 RNA ≥1000 copies/mL after ≥5 months of ART. The evaluable population included 178 participants from a randomly selected subcohort (16 with VF, 162 without VF) and 83 additional participants with VF. In the subcohort, 16% of participants harboured ≥1 majority DRM. The presence of any majority DRM was associated with a 3-fold greater risk of VF (P = 0.002), which increased to 9.2-fold (P < 0.001) in those with <2 active drugs. Thirteen percent of participants harboured MV DRMs in the absence of majority DRMs. Presence of MVs alone had no significant impact on the risk of VF. Inclusion of pre-ART MVs with majority DRMs improved the sensitivity but reduced the specificity of predicting VF. In a South African cohort, the presence of majority DRMs increased the risk of VF, especially for participants receiving <2 active drugs. The detection of drug-resistant MVs alone did not predict an increased risk of VF, but their inclusion with majority DRMs affected the sensitivity/specificity of predicting VF.

Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

BackgroundCopy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs.MethodsA multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.ResultsThere were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated.ConclusionsThe developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

Molecular surveillance of pfcrt, pfmdr1 and pfk13-propeller mutations in Plasmodium falciparum isolates imported from Africa to China

BackgroundThe emergence and spread of multidrug resistance poses a significant risk to malaria control and eradication goals in the world. There has been no indigenous malaria cases reported in China since 2017, and China is approaching national malaria elimination. Therefore, anti-malarial drug resistance surveillance and tracking the emergence and spread of imported drug-resistant malaria cases will be necessary in a post-elimination phase in China.MethodsDried blood spots were obtained from Plasmodium falciparum-infected cases returned from Africa to China between 2012 and 2015, prior to anti-malarial drug treatment. Whole DNA were extracted and known polymorphisms relating to drug resistance of pfcrt, pfmdr1 gene, and the propeller domain of pfk13 were evaluated by nested PCR and sequencing. The haplotypes and prevalence of these three genes were evaluated separately. Chi-squared test and Fisher's exact test were used to evaluate differences among the different sub-regions of Africa. A P value < 0.05 was used to evaluate differences with statistical significance. The maps were created using ArcGIS.ResultsA total of 731 P. falciparum isolates were sequenced at the pfcrt locus. The wild type CVMNK was the most prevalent haplotype with prevalence of 62.8% and 29.8% of the isolates showed the triple mutant haplotype CVIET. A total of 434 P. falciparum isolates were successfully sequenced and pfmdr1 allelic variants were observed in only codons 86, 184 and 1246. Twelve haplotypes were identified and the prevalence of the wild type pfmdr1 NYD was 44.1%. The single mutant pfmdr1 in codons 86 and 184 was predominant but the haplotype NYY with single mutation in codon 1246 was not observed. The double mutant haplotype YFD was common in Africa. About 1,357 isolates were successfully sequenced of pfk13-propeller domain, the wild type was found in 1,308 samples (96.4%) whereby 49 samples (3.6%) had mutation in pfk13. Of 49 samples with pfk13 mutations, 22 non-synonymous and 4 synonymous polymorphic sites were confirmed. The A578S was the most common mutation in pfk13-propeller domain and three mutations associated with artemisinin resistance (M476I, R539T, P553L) were identified in three isolates.ConclusionThis study provides evidence that could give insight into potential issues with anti-malarial drug resistance to inform national drug policy in China in order to treat imported cases.

Prevalence and molecular characterizations of seven additional drug resistance among multidrug-resistant tuberculosis in China: A subsequent study of a national survey

The drug resistance prevalence data facilitates selection of the initial drug for treating multidrug-resistant tuberculosis (MDR-TB). The aim of this study was to investigate the prevalence and molecular characterization of seven additional types of drug resistances among MDR-TB isolates collected from the first/only nationwide drug resistance surveillance in China. A total of 391 out of the 401 MDR-TB strains were successfully recovered by Löwenstein-Jensen medium. Drug susceptibility testing was performed against moxifloxacin (Mfx), bedaquiline (Bdq), linezolid (Lzd), clofazimine (Cfz), cycloserine (Cs), delamanid (Dlm) and pyrazinamide (PZA). The strains were subjected to whole-genome sequencing for the analysis corresponding drug resistant genes and their profiles. 269 (68.80%) were simple MDR-TB, 28 (7.16%) were extensively drug-resistant tuberculosis (XDR-TB) and 94 (24.04%) were pre-XDR-TB. Dlm, Lzd, Cfz and Bdq presented the lowest drug resistant rates i.e. 3.32% (13/391), 3.84% (15/391),6.65% (26/391) and 7.16% (28/391), respectively. Mfx (17.39%, 68/391) and CS (13.55%, 53/391) also demonstrated strong potencies against the MDR strains, whereas PZA (38.36%, 150/391) presented much higher resistant rate. 54.41% (37/68) Mfx-resistant strains carried mutations located within gyrA or gyrB. 70.15% (94/134) PZA-resistant strains had pncA mutations. Two of the 26 Cfz-resistant isolates had mutation in Rv0678 were also resistant to Bdq. Dlm, Lzd, Cfz and Bdq exhibited excellent activity against MDR-TB, including XDR-TB. These data highlighted the necessity of a timely, feasible and reliable DST, while genotypic DST for Mfx and PZA is promising at this moment.

Longitudinal Study of the Drug Resistance in Klebsiella pneumoniae of a Tertiary Hospital, China: Phenotypic Epidemiology Analysis (2013-2018).

PurposeMulti-drug resistant Klebsiella pneumoniae (MDR KP) is spreading worldwide and has posed a huge medical burden to public health. However, studies on drug resistance surveillance of KP, especially MDR KP, with a large longitudinal sample size in a tertiary hospital are rare. This study aims to investigate phenotypic epidemiology characteristics of 4128 KP isolates in a Chinese tertiary hospital covering a period of 5 years.MethodsAll the KP clinical isolates were retrospectively collected from a tertiary hospital in Hunan province of China from Jan 5, 2013 to Jul 24, 2018. All the isolates were identified by MALDI-TOF MS analysis. Twenty-four antimicrobial agents were tested by antimicrobial susceptibility testing. Fisher exact test and logistic regression were used to analyze the association between clinical factors and antimicrobial non-susceptibility for seven second-choice antimicrobials.ResultsA total of 4128 KP isolates were collected in our study. The non-susceptible rates (NSRs) to ertapenem, imipenem and tigecycline increased considerably from 2013 to 2018 (13.6% to 28.6%, 10.1% to 28.9%, 10.8% to 46.5%, respectively). Amikacin presents the lowest NSR among 3 aminoglycosides (3.8–22.8%). The multi-drug NSRs among KP isolates to second-choice antimicrobials (88.6–100%) were higher than to all drugs (68.0%). The NSRs varied significantly among departments and sample sources. Higher ETP/IPM/AK NSRs (39.8/39.7/30.6%) were observed in Intensive Care Unit, and ETP/IPM non-susceptible isolates tended to distribute in cerebrospinal fluid. From 2015 to 2017, the NSRs of ETP, IPM, and AK showed an opposite trend of seasonal fluctuations to SXT.ConclusionHigher multi-drug resistance (MDR) rates were observed in KP isolates to second-choice antimicrobials than to others, among which MDR rates to carbapenems or AK are the highest. A unique pattern of MIC and time distributions of MDR were observed. Clinical factors including gender were correlated with MDR rates of KP. Isolates in ICU and CSF showed higher NSRs in carbapenems which should be paid more attention to, and temporal distribution of NSRs was observed.

Microdrop Human Immunodeficiency Virus Sequencing for Incidence and Drug Resistance Surveillance.

Precise and cost-efficient human immunodeficiency virus (HIV) incidence and drug resistance surveillances are in high demand for the advancement of the 90-90-90 "treatment for all" target. We developed microdrop HIV sequencing for the HIV incidence and drug resistance assay (HIDA), a single-blood-draw surveillance tool for incidence and drug resistance mutation (DRM) detection. We amplified full-length HIV envelope and pol gene sequences within microdroplets, and this compartmental amplification with long-read high-throughput sequencing enabled us to recover multiple unique sequences. We achieved greater precision in determining the stage of infection than current incidence assays, with a 1.2% false recency rate (proportion of misclassified chronic infections) and a 262-day mean duration of recent infection (average time span of recent infection classification) from 83 recently infected and 81 chronically infected individuals. Microdrop HIV sequencing demonstrated an increased capacity to detect minority variants and linked DRMs. By screening all 93 World Health Organization surveillance DRMs, we detected 6 pretreatment drug resistance mutations with 2.6%-13.2% prevalence and cross-linked mutations. HIDA with microdrop HIV sequencing may promote global HIV real-time surveillance by serving as a precise and high-throughput cross-sectional survey tool that can be generalized for surveillance of other pathogens.

Mutation of Gyra Gene Found In Mycobacterium Leprae From Leprosy Patient In West Papua and Papua, Indonesia

Cases of leprosy in Indonesia are still high, especially in the provinces of West Papua, North Maluku and Papua. Drug resistance surveillance and typing strains of Mycobacterium leprae are useful molecular tools for leprosy control especially in the three Provinces. The purpose of this study was to identify mutations in the gyrA M. leprae gene obtained from leprosy patients in the provinces of West Papua and Papua on a molecular basis. M. leprae samples obtained from leprosy patients were extracted and continued with PCR and sequencing in the M. leprae gyrA gene. The sequencing results are aligned with M. leprae TN sequences to identify mutations. The phylogenetic tree was constructed using Mega 7 to get the M. leprae gyrA cluster. The RNAalifold server was employed to generate the conserved 2D structure for the gyrA MSAs. Six variants were found in the gyrA M. leprae obtained from the provinces of West Papua and Papua. The six variants are H71R, K73R, D95G, A101T, R107W, A127V. The existence of mutations in the gyrA M. leprae gene found in this study can be information in the treatment of leprosy in Papua if using Ofloxacin as an alternative treatment. Based on phylogenetic analysis found there are three distinct clusters of gyrA gene. The five variants are H71R, K73R, A101T, R107W, A127V are new variant of gyrA M. leprae. The D95G variant has been confirmed to cause resistance to Fluoroquinolone by in vitro methods, while the H71R, K73R, A101T, R107W, A127V variants are new variants whose effects on the fluoroquinolone are unknown. Thus, further analysis is needed to study the effects of the five variants on ofloxacin.