Threefold Increase in the Number of Drug Resistant TB Cases after Introduction of Universal Drug Susceptibility Testing: Experiences from Two South India Districts

Background: In India, tuberculosis (TB) is a major public health problem, and the advent of drug resistance TB (DR-TB) has worsened the situation. The Revised National TB Control Programme (RNTCP) has introduced universal drug susceptibility testing (UDST) for all diagnosed TB cases in 2018. We conducted this study to know the advantage of implementing UDST when compared to selective testing existent in 2017 on key diagnostic cascade parameters and to identify the challenges in the implementation of UDST. Methods: The study was conducted in two districts of Karnataka, India during January 2017-December 2018. The quantitative part consisted of before-and-after design and the qualitative part consisted of descriptive design. Results: In 2017 (during selective testing/“before” period) out of the 2440 TB patients, 80 (3%) were diagnosed with Isoniazid and Rifampicin resistance patients;in contrast in 2018 (during UDST/“after” period) of the 5129 TB patients 258 (5%) were diagnosed with Isoniazid and Rifampicin resistance. However, the proportion of eligible patients tested for rifampicin resistance during the “after” period was 60% when compared to 100% during the “before” period and median turnaround time for testing was also longer during the “after” period when compared to the “before” period (32.5 days vs 27.5 days). Major reasons for these two gaps were found to be difficulties in collecting sputum specimens and transportation. Conclusion: The rollout of UDST has led to a three-fold increase in a number of DR-TB cases detected in the region. There is a need for the programme to increase the proportion tested for DST by increasing the laboratory capacity and address the challenges in sputum collection and transportation.

Drug Susceptibility Test of Volatile Oil of Artemisiaargyi to Avian Pathogenic Escherichia coli

The volatile oil of Artemisia argyi was extracted by ultrasonic assisted extraction, and the extraction rate of volatile oil was 0.68%. Thevolatile oil of A. argyi was emulsified with 1% Tween-80, and drug susceptibility test was conducted with avian Escherichia coli. The results showedthat the volatile oil of A. argyi had antibacterial effect against avian E. coli, and the minimal inhibitory concentration was 50 mg/mL. Taking sixcommon antibiotics as the control, drug susceptibility test was conducted with volatile oil of A. argyi. The results showed that 10 strains of E. coliwere sensitive to the volatile oil of A. argyi, three of which had different degrees of resistance and one had the tendency of resistance.

Performance of the microscopic observation drug susceptibility assay in pyrazinamide susceptibility testing for Mycobacterium tuberculosis

Background Drug susceptibility assay is very important in tuberculosis therapy. Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis （M. tuberculosis） is difficult and time consuming by conventional methods. In this study, we aimed to evaluate the performance of the microscopic observation drug susceptibility （MODS） assay in the detection of pyrazinamide resistance in M. tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen （L J） proportion method. Methods M. tuberculosis clinical isolates （n=132） were tested by the MODS and the Wayne assay： the results were compared with those obtained by the LJ proportion method. Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods. Results Compared to the LJ results, the sensitivity and specificity of the MODS assay were 97.8% and 96.5% respectively; the sensitivity and specificity of the Wayne assay were 87.0% and 97.7% respectively. Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant （3 tests）, in 1 of the 4 strains （LJ only）, in 42 of 48 strains （at least I test）, but no mutations in 1 strain sensitive according to the MODS assay only. The MODS assay, Wayne assay and LJ proportion method provided results in a median time of 6, 7 and 26 days respectively. Conclusions MODS assay offers a rapid, simple and reliable method for the detection of pyrazinamide resistance in M. tuberculosis and is an optimal alternative method in resource limited countries.

Isolation,identification,and virulence gene analysis of pathogenic Aeromonas dhakensis in Macrobrachium rosenbergii and histopathological observation

To identify the cause of mass mortality of adult Macrobrachium rosenbergii in a farm in Gaoyou City,Jiangsu Province,China,a dominant strain named DKQ-1 was isolated from the hepatopancreas of dying M.rosenbergii and identified as Aeromonas dhakensis by purification culture,biochemical characterization,and 16S rRNA and gyrB gene sequence analysis.The results of the challenge test revealed that the strain was highly pathogenic and the 50%lethal dose(LD_(50))in 72 h to M.rosenbergii was 1.54×10^(5)CFU/mL.The amplification results of virulence genes show that strain DKQ-1 carried 9 virulence genes,including ascV,aexT,aer,act,lip,ompAI,gcaT,acg,and exu,supporting the strong virulence of strain DKQ-1 to M.rosenbergii.Histopathological observation of the hepatopancreas,gills,and intestines indicated that DKQ-1 injection into M.rosenbergii could cause serious tissue damage,which further supported the strong virulence of this strain.In addition,a drug susceptibility test revealed that strain DKQ-1 was sensitive to 16 kinds of antibiotics,resistant to 9 kinds of antibiotics,and had intermediate resistance to spectinomycin and kanamycin.This study is the first report of A.dhakensis isolated from M.rosenbergii and provided a reference for the pathogen identification of bacterial diseases in M.rosenbergii,and for the prevention and treatment caused by A.dhakensis.

Susceptibility of Mixed Infection of Ureaplasma Urealyticum and Mycoplasma Hominis to Seven Antimicrobial Agents and Comparison with that of Ureaplasma Urealyticum Infection

In order to investigate the susceptibility of mixed infection of Ureaplasma Urealyticum (UU) and Mycoplasma Hominis (MH) to 7 kinds of antimicrobial agents and comparison with that of UU infection in NGU patients, the in vitro susceptibility was determined by using microdilution method. The positive results were analyzed. The results showed that the sequence of susceptibility to 7 kinds of antimicrobial agents for both UU infection group and UU-MH mixed infection group was almost the same from the highest susceptibility to the lowest accordingly: Josamycin, Doxycycline, Minocycline, Sparfloxacin, Roxithromycin, Ofloxacin and Azithromycin. The total drug resistance rate for UU-MH mixed infection group (97. 67 %) was significantly higher than that for UU infection group (44. 67 %, P<.0. 01). The highest drug resistance rate in UU group and UU-MH mixed infection group was 31. 33 % (Ofloxacin) and 90. 48 % (Azithromycin) respectively. UU-MH mixed infection showed an increased drug resistance and changes of drug resistance spectrum.

Prevalence and Anti-Microbial Susceptibility of Methicillin Resistant Staphylococcus aureus at Moi Teaching and Referral Hospital Eldoret

Background: Methicillin-resistant Staphylococcus aureus (MRSA) are pathogens that have major negative impact in all aspects of patient care and are increasingly being recognized as troublesome pathogens in the community. The MRSA are multi-drug resistant bacteria responsible for higher morbidity, mortality and cost of treatment in the affected patients. Research on MRSA antimicrobial susceptibility and resistance in developing countries is limited;hence accurate burden is not certain. Objective: To determine the prevalence and susceptibility pattern of MRSA in S. aureus isolates from patients treated in various units at Moi Teaching and Referral Hospital. Study Design: Cross-sectional design was used to conduct the study. Methods and Materials: Staphylococcus aureus stored isolates from patients at various units at Moi Teaching and Referral Hospital (MTRH) were identified by laboratory conventional methods. Methicillin resistant Staphylococcus aureus was determined using cefoxitin and oxacillin, discs and confirmed by Penicillin Binding Protein (PBP2a’) latex agglutination test. Susceptibility testing was performed according to the Clinical Laboratory Standards Institute procedures. Control strains used were: ATCC 43300 for Methicillin resistant S. aureus (MRSA) strains and ATCC 29213 for methicillin susceptible S. aureus (MSSA) strains. Nine anti-microbials (Erythromycin, Gentamycin, Tetracycline, Vancomycin, Rifampin, Linezolid, Ciprofloxacin, Clindamycin and Fucidicacid) were used to test the anti-microbial susceptibility patterns. Control organisms were run along with the isolates for quality Assurance. The data was analyzed using STATA version 11. Categorical variables were summarized as frequencies and the corresponding percentages. Results were presented using tables and graphs. Results: A total of 107 isolates of S. aureus were obtained, of which 39 (37%) were MRSA. Most of the MRSA, 13 (33%) and 7 (17%), were found in pus and tracheal aspirate samples respectively. Majority of the MRSA isolates were from surgical wards and intensive care unit. The MRSA isolates were highly resistant to erythromycin (92%;36/39) and tetracycline (92% 36/39) and moderately susceptible to linezolid (77% 30/39), Vancomycin (75% 29/39) and fucidic acid (67% 26/39). A total of 28 (74.4%) MRSA isolates were Clindamycin inducible resistant. Conclusions and Recommendations: This study showed that MRSA is a significant pathogen at MTRH. The MRSA were most detected in pus specimens and least in peritoneal aspirates. Routine Screening of S. aureus isolates for MRSA strains at MTRH is therefore imperative. Infection control measures should be put in place to reduce prevalence and prevent the spread of MRSA at MTRH.

Risk Factors for Group B <i>Streptococcus</i>Colonization and Drugs Sensitivity Pattern in a Nigerian Obstetric Population

Background: Group B Streptococcus (GBS) is a major cause of bacterial infections in the perinatal period, of which colonization prevalence among Northern-Nigerian pregnant women is scarce. We attempted to determine 1) its prevalence, 2) risk factors for GBS colonization and 3) drugs-susceptibility. Methodology: This cross-sectional study involved 185 pregnant women between 35 - 37 weeks of gestation at tertiary health center of Sokoto, Nigeria. Vaginal/rectal swabs were collected, were cultured for GBS and tested for drug-susceptibilities. The study was conducted between December, 2017 and April, 2018. Results: One hundred and eighty five (185) pregnant women participated in this study. GBS vaginal-colonization-rate was 3.8% (7/185). A significance relationship was observed between GBS-colonization and socio-economic class, as 57.10% (4/7) of the GBS positive women were of low-socio economic class (p 0.035). No associations were observed between GBS-colonization and the followings: maternal age, parity, poor obstetric outcome-history. All the 7 GBS positive cultures were sensitive to Clindamycin. One was sensitive to both Clindamycin and Ceftriaxone. None was sensitive to Penicillin. Conclusion: The prevalence of GBS colonization was low in this area. Maternal socio-economic class is found to be a risk of GBS-colonization.

Isolation and Identification of Bacterial Pathogens of Proteus vulgaris and Chryseobacterium meningosepticum from Peodiscus sinensis and Observation on the Histopathology

Two dominant strains of bacteria Lb18-01 and Lb18-02 were isolated and purified from scabies,liver and intestine of diseased Peodiscus sinensis.By artificial infection test,the two strains were verified as pathogenic strains with similar characters to that of natural infectious cases.The strains Lb18-01 and Lb18-02 showed strong pathogenicity to healthy P.sinensis in the artificial infection experiment,so they were the pathogenic strain of the disease.According to the morphology,physicochemical characteristics,16 S rDNA sequence analysis and phylogenetic tree clustering,the pathogenic strains Lb18-01 and Lb18-02 were identified as Proteus vulgaris and Chryseobacterium meningosepticum which were tolerant to 20 drugs such as penicillin,tetracycline and ampicillin.Histopathological observation on diseased P.sinensis showed the pathological symptoms of sepsis such as hemorrhage and congestion of liver,spleen and intestine,and glomerular disintegration.

Faropenem Susceptibility of Multidrug-resistant Contemporary Clinical Isolates from Zhejiang Province, China

Alternative antimicrobial therapies are urgently needed for a variety of multidrug-resistant bacterial pathogens.Faropenem is an orally available b-lactam in the class of carbapenem antibiotics.Thus far,little information is available on faropenem susceptibility of multidrug-resistant clinical isolates from China.Therefore,in the current study 141 contemporary clinical isolates,including methicillin-resistant Staphylococcus aureus(MRSA:n=18),coagulase-negative staphylococci(CNS:n=16),Enterococcus faecalis(n=14),Enterococcus faecium(n=7),Pseudomonas aeruginosa(n=25),Klebsiella pneumoniae(n=21),Escherichia coli(n=20),and Acinetobacter baumannii(n=20),were collected between March 2018 and March 2019 and tested for susceptibility to faropenem and other antimicrobials using the broth microdilution method.All E.faecium,P.aeruginosa,and A.baumannii isolates were fully resistant to faropenem.However,most of the MRSA,CNS,E.faecalis,K.pneumonia,and E.coli isolates appeared to be susceptible to faropenem,with minimal inhibitory concentration90 values of 4mg/L,2mg/L,2mg/L,4mg/L,and 2mg/L,respectively.Therefore,faropenem could be considered as alternative therapy for future treatment of multidrug-resistant MRSA,CNS,E.faecalis,K.pneumonia,and E.coli infections in China.

White-opaque Switching in Different Mating Type-like Locus Gene Types of Clinical Candida albicans Isolates

Background：Candida albicans （C.albicans） can become a pathogen causing superficial as well as life-threatening systemic infections,especially in immunocompromised patients.Many phenotypic attributes contribute to its capacity to colonize human organs.In our study,93 C.albicans isolates from patients of various candidiasis in a hospital of China were surveyed.We aimed to investigate the white-opaque （WO） switching competence,drug sensitivity,and virulence of mating type-like （MTL） a/α isolates.Methods：Internal transcribed spacer （ITS） gene and the MTL configuration were detected in all the isolates by reverse transcription-polymerase chain reaction.White/opaque phenotype and doubling time of cell growth were determined.The minimum inhibitory concentrations of antifungal agent were measured using broth microdilution method.Results：Sixty-four isolates （69.6%） were classified to serotype A,19 （20.6%） to serotype B,and 9 （9.8%） to serotype C.Moreover,phylogenetic analysis showed that these isolates were divided into four different subgroups of ITS genotypes.Most of our clinical isolates were MTLa/α type,while 6.8% remained MTLa or MTLα type.The frequency of opaque phenotype was 71.0% （66 isolates）.Following the guidelines of Clinical and Laboratory Standards Institute M27-A3,all isolates were susceptible to caspofungin and a few （0.6-3.2%） of them showed resistance against amphotericin B,flucytosine,fluconazole,itraconazole,and voriconazole.Conclusions：From these analyses,there were comparatively more C.albicans strains classified into serotype B,and the frequency of opaque phase strains was significant in the clinical isolates from China.Genetic,phenotypic,or drug susceptibility patterns were not significantly different from previous studies.MTLa/α isolates could also undergo WO switching which facilitates their survival.