Clinical Characteristics and Treatment Outcomes for Patients Infected with Mycobacterium haemophilum.

Mycobacterium haemophilum is a nontuberculous mycobacterium that can infect immunocompromised patients. Because of special conditions required for its culture, this bacterium is rarely reported and there are scarce data for long-term outcomes. We conducted a retrospective study at Siriraj Hospital, Bangkok, Thailand, during January 2012-September 2017. We studied 21 patients for which HIV infection was the most common concurrent condition. The most common organ involvement was skin and soft tissue (60%). Combination therapy with macrolides and fluoroquinolones resulted in a 60% cure rate for cutaneous infection; adding rifampin as a third drug for more severe cases resulted in modest (66%) cure rate. Efficacy of medical therapy in cutaneous, musculoskeletal, and ocular diseases was 80%, 50%, and 50%, respectively. All patients with central nervous system involvement showed treatment failures. Infections with M. haemophilum in HIV-infected patients were more likely to have central nervous system involvement and tended to have disseminated infections and less favorable outcomes.

d-Cycloserine Pharmacokinetics/Pharmacodynamics, Susceptibility, and Dosing Implications in Multidrug-resistant Tuberculosis: A Faustian Deal.

Background: d-cycloserine is used to treat multidrug-resistant tuberculosis. Its efficacy, contribution in combination therapy, and best clinical dose are unclear, also data on the d-cycloserine minimum inhibitory concentration (MIC) distributions is scant. Methods: We performed a systematic search to identify pharmacokinetic and pharmacodynamic studies performed with d-cycloserine. We then performed a combined exposure-effect and dose fractionation study of d-cycloserine in the hollow fiber system model of tuberculosis (HFS-TB). In parallel, we identified d-cycloserine MICs in 415 clinical Mycobacterium tuberculosis (Mtb) isolates from patients. We utilized these results, including intracavitary concentrations, to identify the clinical dose that would be able to achieve or exceed target exposures in 10000 patients using Monte Carlo experiments (MCEs). Results: There were no published d-cycloserine pharmacokinetics/pharmacodynamics studies identified. Therefore, we performed new HFS-TB experiments. Cyloserine killed 6.3 log10 colony-forming units (CFU)/mL extracellular bacilli over 28 days. Efficacy was driven by the percentage of time concentration persisted above MIC (%TMIC), with 1.0 log10 CFU/mL kill achieved by %TMIC = 30% (target exposure). The tentative epidemiological cutoff value with the Sensititre MYCOTB assay was 64 mg/L. In MCEs, 750 mg twice daily achieved target exposure in lung cavities of 92% of patients whereas 500 mg twice daily achieved target exposure in 85% of patients with meningitis. The proposed MCE-derived clinical susceptibility breakpoint at the proposed doses was 64 mg/L. Conclusions: Cycloserine is cidal against Mtb. The susceptibility breakpoint is 64 mg/L. However, the doses likely to achieve the cidality in patients are high, and could be neurotoxic.

Epidemiology, Clinical Characteristics, Sites of Infection and Treatment Outcomes of Mucocutaneous Candidiasis Caused by Non-Albicans Species of Candida at a Dermatologic Clinic.

BACKGROUND: Increasing numbers of mucocutaneous infection due to non-albicans species of Candida (N-CA) had been reported. Laboratory based studies showed multidrug resistance in N-CA population. OBJECTIVE: Demonstrate epidemiology, clinical characteristics, sites of infection, and treatment outcomes of mucocutaneous candidiasis caused by N-CA at a dermatologic clinic, including statistical evaluation data between N-CA and C. albicans infections. MATERIAL AND METHOD: This was a cross sectional study of outpatients with mucocutaneous infection due to Candida at Dermatologic clinic between January 2012 and June 2014. Vaginal candidiasis was excluded. Demographic, clinical, laboratory data, and treatment outcomes were collected. RESULTS: Among 760 patients presented with mucocutaneous candidiasis, 307 (40.4%) were infected with N-CA. The mean age (SD) of N-CA patients was 63.6 (10.4) years and 74.6% were female. The majority of N-CA cases were isolated from patients' nails (n = 293, 95.4%) while eight (2.6%) were detected from their skin, and six (2%)from oral mucosa. Comparison between N-CA and C. albicans, skin, and mucosa infection were significantly demonstrated in C. albicans groups (p < 0.001). Among nail infected patients, C. albicans infections had significant higher severity than the N-CA infection (p = 0.017). Median time to cure in N-CA population was 169 days, which had no significant difference from C. albicans groups (211 days, p = 0.499). CONCLUSION: Forty percent of mucocutaneous candidiasis was caused by N-CA. Nails were the most common sites of N-CA infections but N-CA was sometime found in skin and mucosa. Treatment outcomes of N-CA population were not significantly different from those of C. albicans groups.

PREVALENCE AND FACTORS ASSOCIATED WITH MULTIDRUG-RESISTANT TUBERCULOSIS AT SIRIRAJ HOSPITAL, BANGKOK, THAILAND.

The objective of this study was to determine the prevalence and factors associated with multidrug-resistant tuberculosis (MDR-TB) at Siriraj Hospital, Bangkok, Thailand. We conducted a retrospective unmatched case-control study of patients clinically diagnosed and microbiologically confirmed to have tuber- culosis (TB) at Siriraj Hospital from 2010 to 2012. Patient characteristics, clinical data, microbiological findings, outcomes and drug susceptibilities were recorded. A total of 188 subjects were included in the study; 52.1% (98) were males; the mean age was 48.9 years. Subjects were categorized into one of two groups, as follows: non-MDR-TB (141 patients) and MDR-TB (47 patients). The prevalence of MDR- TB was 2.6%. Co-morbidities of study subjects included diabetes mellitus (16.5%), HIV infection (16%) and cancer (5.9%). One hundred thirty-one patients (69.7%) had pulmonary TB. Factors significantly associated with MDR-TB were age < 65 years (OR = 6.94; 95% CI: 1.02-45.49; p = 0.048), history of TB (OR = 51.86; 95% CI: 12.35-217.79; p < 0.001), HIV co-infection (OR = 3.83; 95% CI: 1.02-14.38; p = 0.047) and alcohol consumption (OR = 3.90; 95% CI: 1.03-14.72; p = 0.045). Of the 146 patients for whom a clinical outcome was available, 51 (34.9%) had an unfavorable outcome. Poor compliance (OR = 13.51; 95% CI: 3.97-45.45; p < 0.001) and previous history of TB (OR = 8.16; 95% CI: 1.76-37.73; p = 0.007) were associated with an unfavorable outcome. MDR-TB was significantly associated with: patients aged < 65 years, those with a previous history of TB, those with HIV co-infection and those who drank alcohol. These factors should be kept in mind when treating TB patients at Siriraj Hospital, Thailand.

Use of mycobacteriophage quantitative PCR on MGIT broths for a rapid tuberculosis antibiogram.

Phenotypic culture-based drug susceptibility testing (DST) for Mycobacterium tuberculosis is a valuable tool to identify four to six active drugs for individualized multidrug-resistant (MDR) tuberculosis (TB) regimens. Current culture-based methods are slow, however; therefore, we evaluated a rapid mycobacteriophage-based quantitative PCR (qPCR) assay for use directly on M. tuberculosis-positive MGIT broths. We compared phage qPCRs, using a simple cutoff of 3 for the ΔCq value (where Cq is quantification cycle, and ΔCq is calculated as the Cq of starting phage minus the Cq of TB isolates in drug-containing medium), on 325 clinical M. tuberculosis MGIT broth cultures versus the respective subcultured isolates tested by agar proportion. The median accuracy for the 13 drugs/concentrations tested was 98%, with most discrepancies being false-resistant results. Evaluation of phage qPCR on greater numbers of resistant strains of 393 isolates grown on Löwenstein-Jensen medium showed similar findings, with a median accuracy, sensitivity, and specificity of 97%, 90%, and 99%, respectively. This rapid culture-based DST methodology can be performed for any drug on TB-positive MGIT broths, with a specimen-to-antibiogram turnaround time of approximately 23.9 days, compared with waiting 58.6 days for isolate growth on solid medium followed by agar proportion DST.

Multi-probe real-time PCR identification of four common Candida species in blood culture broth.

We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.

Clinical features and outcomes of isoniazid mono-resistant pulmonary tuberculosis.

OBJECTIVE: To determine the characteristics of pulmonary tuberculosis (TB) patients harbored organisms with isoniazid mono-resistant drug susceptibility pattern. MATERIAL AND METHOD: A retrospective review of medical records for all culture-proven adult pulmonary TB patients in Siriraj Hospital between July 2009 and July 2011 was conducted. Demographic data, clinical presentations, and radiological characteristics were recorded and compared between isoniazid mono-resistant and other-resistant groups. Treatment regimens with outcome determination of patients infected with isoniazid mono-resistant strains were also verified. RESULTS: Among 489 patients during the present study period, 28 were infected with isoniazid mono-resistant strain (5.7%). The mean age was 53 +/- 18 years, and 8% of them had a history of previous treatment in the past. When compared with those infected with any other form of resistant strains, isoniazid mono-resistant pulmonary TB patients tended to have less radiographic cavitary lesion (8.3% vs. 26.7%, p = 0.006) but no significant difference was seen in term of demographic data and clinical presentations. All of them who had completed the treatment were cured. No difference in cure rate and relapse rate among patients treated with quinolone or non-quinolone containing regimens. CONCLUSION: Isoniazid mono-resistance shares common clinical features with other resistances pulmonary TB, except for less cavitary lesion from initial chest radiograph. Appropriate drug susceptibility testing with prompt regimen adjustment can lead to a favorable treatment outcome.

Rapid detection of pyrazinamide resistant Mycobacterium tuberculosis by high resolution melting curve analysis.

OBJECTIVE: To develop a high-resolution melting curve analysis (HRM) assay for detection of PZA resistance. MATERIAL AND METHOD: Thirty samples of PZA-susceptible M. tuberculosis and eight isolates of PZA-resistant M. tuberculosis were included in the experiment. Five sets of primers were designed to cover the pncA gene and its upstream nucleotides. The pncA gene fragments were amplified by the PCR method. Determination of pncA mutation in the sample by comparing their melting behavior of the PCR products with the M. tuberculosis wild type by using Gene scanning software of the LightCycler 480 instrument. RESULTS: Mutations were clearly detected in all PZA resistant samples by the HRM, whereas all PZA susceptible samples showed no mutation in the pncA gene. Results were concordant with the drug susceptibility testing by using BACTEC MGIT 960 PZA kit and mutation detection by the DNA sequencing method. CONCLUSION: This HRM method offers a rapid and reliable screen for PZA resistant M. tuberculosis.

Prosthetic valve endocarditis caused by Histoplasma capsulatum: the first case report in Thailand.

The authors report a rare case of fungal endocarditis caused by Histoplasma capsulatum in an immunocompetent woman with mitral valve prosthesis. The patient presented with chronic fever and embolic phenomenon. Transthoracic and transesophageal echocardiography revealed a mobile mass attached to mitral prosthetic valve and her blood cultures were negative for both bacteria and fungi. The diagnosis was made by presence of budding yeasts in the histopathological findings of the vegetation and recovery of H. capsulatum from tissue culture of the excised vegetation. The patient was improved after a 6-week course of amphotericin B. Fungal endocarditis caused by Histoplama capsulatum is rare but should be considered as a possible causative organism in culture-negative endocarditis. To our knowledge, this is the first case report of H. capsulatum endocarditis in Thailand.

Nanopore Sequencing Discloses Compositional Quality of Commercial Probiotic Feed Supplements.

The market for the application of probiotics as a livestock health improvement supplement has increased in recent years. However, most of the available products are quality-controlled using low-resolution techniques and un-curated databases, resulting in misidentification and incorrect product labels. In this work, we deployed two workflows and compared results obtained by full-length 16S rRNA genes (16S) and metagenomic (Meta) data to investigate their reliability for the microbial composition of both liquid and solid forms of animal probiotic products using Oxford Nanopore long-read-only (without short-read). Our result revealed that 16S amplicon data permits to detect the bacterial microbiota even with the low abundance in the samples. Moreover, the 16S approach has the potential to provide species-level resolution for prokaryotes but not for assessing yeast communities. Whereas, Meta data has more power to recover of high-quality metagenome-assembled genomes that enables detailed exploration of both bacterial and yeast populations, as well as antimicrobial resistance genes, and functional genes in the population. Our findings clearly demonstrate that implementing these workflows with long-read-only monitoring could be applied to assessing the quality and safety of probiotic products for animals and evaluating the quality of probiotic products on the market. This would benefit the sustained growth of the livestock probiotic industry.

Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis.

Managing drug-resistant Mycobacterium tuberculosis requires drug susceptibility testing, yet conventional drug susceptibility testing is slow, and molecular testing does not yield results for all antituberculous drugs. We addressed these challenges by utilizing real-time PCR of mycobacteriophage D29 DNA to evaluate the drug resistance of clinical M. tuberculosis isolates. Mycobacteriophages infect and replicate in viable bacterial cells faster than bacterial cells replicate and have been used for detection and drug resistance testing for M. tuberculosis either by using reporter cells or phages with engineered reporter constructs. Our primary protocol involved culturing M. tuberculosis isolates for 48 h with and without drugs at critical concentrations, followed by incubation with 10(3) PFU/ml of D29 mycobacteriophage for 24 h and then real-time PCR. Many drugs could be incubated instantly with M. tuberculosis and phage for 24 h alone. The change in phage DNA real-time PCR cycle threshold (C(T)) between control M. tuberculosis and M. tuberculosis treated with drugs was calculated and correlated with conventional agar proportion drug susceptibility results. Specifically, 9 susceptible clinical isolates, 22 multidrug-resistant (MDR), and 1 extensively drug-resistant (XDR) M. tuberculosis strains were used and C(T) control-C(T) drug cutoffs of between +0.3 and -6.0 yielded 422/429 (98%) accurate results for isoniazid, rifampin, streptomycin, ethambutol, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, cycloserine, and linezolid. Moreover, the ΔC(T) values correlated with isolate MIC for most agents. This D29 quantitative PCR assay offers a rapid, accurate, 1- to 3-day phenotypic drug susceptibility test for first- and second-line drugs and may suggest an approximate MIC.

Are two sputum samples enough for diagnosing pulmonary tuberculosis.

OBJECTIVE: To determine the optimum number of sputum specimens for smear and culture in the diagnosis of pulmonary tuberculosis. MATERIAL AND METHOD: A retrospective study was conducted in culture-positive pulmonary tuberculosis patients at Siriraj Hospital during April 2009 to October 2010. Number of sputum specimens and microbiological results were retrieved from the microbiologic laboratory. Positive yield and incremental yield of each sputum specimen were calculated. RESULTS: There were 401 patients during the study period, 153 (38.2%) had positive smear for acid-fast bacilli. Overall diagnostic yields of solid culture media and liquid culture media, were 72.1% and 95.3% respectively. Incremental of overall diagnostic yield from 1 to 2 and 2 to 3 sputum specimens were 8% and 6% respectively. CONCLUSION: In place where a routinely combined smear and culture for every sputum sample submitted to the microbiologic laboratory, two specimens are sufficient for the diagnosis in nearly all pulmonary tuberculosis patients.

Long-Read 16S rRNA Amplicon and Metagenomic Data of Swine Feed-Additive Probiotics Product.

Swine feed-additive probiotics products play a major role in swine performance and welfare by promoting gut health. Here, we present two types of data, including a full-length 16S rRNA amplicon sequence data and a long-read metagenomic sequence data obtained from the same commercial probiotic product.

Full-Length 16S rRNA Gene Amplicon and Metagenome Taxonomic Profiling of Beneficial Microbes in Poultry and Swine Probiotic Product.

Analysis of feed supplements can highlight microbial diversity and the prevalence of antimicrobial resistance (AMR), allowing users to monitor the safety of their animals. The 16S amplicon and metagenomic data generated by nanopore sequencing revealed that Bacillus was the dominant prokaryote, and AMR genes were detected in the animal probiotic products.

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 (C(T)) time values (C(T) control - C(T) drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as ΔCT or as 2(Δ)(CT) 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.

Impact of bronchoalveolar lavage galactomannan on the outcome of patients at risk for invasive pulmonary aspergillosis.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is an important cause of morbidity and mortality among immunocompromised patients especially in neutropenic and patients treated with immunosuppressive drugs. New diagnostic tools have been developed to improve treatment and outcome. Compared with serum galactomannan, bronchoalveolar lavage galactomannan (BAL GM) detection has higher sensitivity (81% vs. 71%) and comparable specificity (87.6% vs. 89%). No study has correlated this test result to clinical outcome. MATERIAL AND METHOD: A prospective non-randomised study was conducted from March to December 2008 in adult patients who were suspected to have invasive pulmonary aspergillosis (IPA). Serum galactomannan levels were measured and bronchoscopy was performed to obtained BAL fluid for direct examination, culture, and measurement of galactomannan level. Response to treatment and mortality within 6-weeks of follow-up were compared between positive and negative BAL GM groups. Factors influencing outcome were also analysed. RESULTS: There were 30 patients with 3 probable, 11 possible and 17 no IPA. Other causative organisms can be identified in 8 of 17 patients in the no IPA group. Overall, BAL GM at the 0.5 cut-off yielded a 46% positive result compared with 13% of serum GM (p = 0.005). There was no significant difference in positive result between BAL GM at 1.0 cut-off and serum GM. By using BAL GM as a mycological criteria, 54% of possible IPA was upgraded to probable IPA. Neither BAL GM nor serum GM results were associated with clinical response and mortality. Recovery of neutropenia was the only factor associated with response to treatment and outcome (p = 0.003). CONCLUSION: BAL GM detection has a higher positive rate than serum GM in patients at risk for IPA. It is helpful in diagnosis and categorization of IPA, but its impact on clinical outcome cannot be demonstrated in this study.

Antimicrobial Resistance in Swine Fecal Specimens Across Different Farm Management Systems.

Antimicrobial use in agricultural animals is known to be associated with increases in antimicrobial resistance. Most prior studies have utilized culture and susceptibility testing of select organisms to document these phenomena. In this study we aimed to detect 66 antimicrobial resistance (AMR) genes for 10 antimicrobial agent classes directly in swine fecal samples using our previously developed antimicrobial resistance TaqMan array card (AMR-TAC) across three different swine farm management systems. This included 38 extensive antimicrobial use (both in treatment and feed), 30 limited antimicrobial use (treatment only), and 30 no antimicrobial use farms. The number of resistance genes detected in extensive antimicrobial use farms was higher than in limited and no antimicrobial use farms (28.2 genes ± 4.2 vs. 24.0 genes ± 4.1 and 22.8 genes ± 3.6, respectively, p < 0.05). A principal component analysis and hierarchical clustering of the AMR gene data showed the extensive use farm samples were disparate from the limited and no antimicrobial use farms. The prevalence of resistance genes in extensive use farms was significantly higher than the other farm categories for 18 resistance genes including bla SHV, bla CTX-M1 group, bla CTX-M9 group, bla VEB, bla CMY2-LAT, aac(6')-lb-cr, qnrB1, gyrA83L-E. coli, armA, rmtB, aac(3)-IIa, mphA, 23S rRNA 2075G-Campylobacter spp., mcr-1, catA1, floR, dfrA5-14, and dfrA17. These genotypic findings were supported by phenotypic susceptibility results on fecal E. coli isolates. To examine the timing of AMR gene abundance in swine farms, we also performed a longitudinal study in pigs. The results showed that AMR prevalence occurred both early, presumably from mothers, as well as after weaning, presumably from the environment. In summary, detection of AMR genes directly in fecal samples can be used to qualitatively and quantitatively monitor AMR in swine farms.

Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth.

Mycobacterium tuberculosis complex, M. avium, and M. intracellulare are the most common causes of systemic bacterial infection in AIDS patients. To identify these mycobacterial isolates in primary blood culture broths, we developed a multiple hybridization probe-based real-time PCR assay using the LightCycler system. The primers were designed to amplify a 320-bp fragment of Mycobacterium 16S rRNA genes. Reaction specificity was evaluated using PCR amplification curves along with specific melting temperatures of probes on DNA extracted from 13 Mycobacterium species. In this study, results showed 100% accuracy for the selected bacterial panel. Detection limits were 350, 600, and 650 colony-forming unit (CFU)/ml blood culture broths for M. tuberculosis complex, M. avium, and M. intracellulare, respectively (1 to 2 CFU/reaction). To evaluate clinical applicability, 341 acid-fast bacilli in blood culture broths were analyzed. In total, 327 (96%) were positively identified: 54.5% M. tuberculosis complex, 37.5% M. avium, and 3.8% M. intracellulare. Results can be available within 3 hours of receiving a broth sample, which makes this rapid and simple assay an attractive diagnostic tool for clinical use.

Clinical outcome and laboratory markers for predicting disease activity in patients with disseminated opportunistic infections associated with anti-interferon-γ autoantibodies.

BACKGROUND: Clinical courses and treatment outcomes are largely unknown in patients with adult-onset immunodeficiency associated with anti-interferon-gamma autoantibodies due to the fact that it was recently recognized and anti-IFN-γ auto-Abs detection is not widely available. METHODS AND FINDINGS: Non-HIV-infected adult patients with detectable anti-IFN-γ auto-Abs diagnosed and followed at Siriraj Hospital, Bangkok, Thailand during January 2013 to November 2016 were prospectively studied. At each follow-up visit, patients were classified as stable or active disease according to symptoms and signs, and all proven OIs were recorded. Laboratory parameters, including erythrocyte sedimentation rate, C-reactive protein, and anti-IFN-γ auto-Abs level, were compared between active and stable disease episodes. We identified 80 patients with this clinical syndrome and followed them up during study period. Seventy-nine patients developed overall 194 proven opportunistic infections. Mycobacterium abscessus (34.5%) and Salmonella spp. (23.2%) were the two most common pathogens identified among these patients. Sixty-three patients were followed for a median of 2.7 years (range 0.6-4.8 years). Eleven (17.5%) patients achieved the drug-free remission period for at least 9 months. Four patients died. Anti-IFN-γ auto-Abs concentration was significantly lower at baseline and decreased over time in the drug-free remission group compared to another group (p = 0.001). C-reactive protein, erythrocyte sedimentation rate and white cell count were found to be useful biomarkers for determining disease activity during follow-up. CONCLUSIONS: Reinfection or relapse of OIs is common despite long-term antimicrobial treatment in patients with anti-IFN-γ auto-Abs. Treatment to modify anti-IFN-γ auto-Abs production may improve long-term outcomes in this patient population.

Diagnosis of feline filariasis assisted by a novel semi-automated microfluidic device in combination with high resolution melting real-time PCR.

BACKGROUND: The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. METHODS: The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were 'spiked' with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. RESULTS: In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi, 15 as Dirofilaria immitis and 7 as| D. repens. CONCLUSIONS: We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.