Molecular epidemiology of Cryptosporidium spp. in an agricultural area of northern Vietnam: A community survey.

The purpose of this study was to investigate the occurrence of Cryptosporidium infection and the potential for transmission of Cryptosporidium spp. between animals and humans in northern Vietnam. A total of 2715 samples (2120 human diarrheal samples, 471 human non-diarrheal samples, and 124 animal stool samples) were collected through our community survey in an agricultural area. All samples were tested for Cryptosporidium spp. by direct immunofluorescence assay (DFA) using a fluorescent microscope. DNA extraction, PCR amplification of three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp. Of 2715 samples, 15 samples (10 diarrheal samples, 2 non-diarrheal samples, and 3 animal stool samples) tested positive by PCR for the COWP gene. Three species of Cryptosporidium spp. were identified as C. canis (from six human diarrheal samples, two human non-diarrheal samples, and one dog sample), C. hominis (from four human diarrheal samples), and C. suis (from two pig samples) by sequencing the amplified COWP and/or SSU-rRNA genes. In terms of C. hominis, the GP60 subtype IeA12G3T3 was detected in all four human diarrheal samples. Although the number of positive samples was very small, our epidemiological data showed that the emerging pattern of each of the three species (C. canis, C. hominis, and C. suis) was different at this study site. While C. hominis and C. suis were only detected in human and pig samples, respectively, C. canis was detected in samples from both dogs and humans. We suspect that C. canis infections in humans at this study site may be due to environmental contamination with animal and human feces.

Molecular epidemiology of Giardia spp. in northern Vietnam: Potential transmission between animals and humans.

Giardia spp. is detected frequently in humans and animals. Although many studies have been conducted on the epidemiology of giardiasis, there is a scarcity of information on the genetic diversity and the dynamics of transmission of Giardia spp. in Vietnam. The zoonotic potential of Giardia spp. remains elusive. The objective of this study was to determine the genetic diversity of Giardia spp. in both humans and livestock to assess the existence of a route of infection between livestock and humans. Our goal was to assess the role animals play in the epidemiology of human infection in northern Vietnam. In Hien Khanh commune in northern Vietnam, 311 households with 1508 residents were randomly selected for a diarrheal cohort study. Of these, 2120 human diarrheal samples were collected from 1508 residents in 2014 and 2017. Of these, non-diarrheal samples were cross-sectionally collected from 471 residents. At the same site, livestock samples from buffalo, dairy and beef cattle, pigs, and dogs were collected. All stool samples were examined for Giardia spp. by Direct Immunofluorescence Assay (DFA) using fluorescent microscope. DNA extraction, PCR analysis of the 3 genes (bg, gdh, tpi), and sequencing analysis were continuously carried out. A total of 23 animal stool samples, 8 human non-diarrheal samples, and 36 human diarrheal samples were Giardia spp. were positive by PCR using the bg and gdh genes. Giardia spp. assemblage AII and E were detected in both animal samples and human samples in this study site. The detection of assemblage E in human stool samples suggests the first human case report in Vietnam. We assume that the unexpected human infection of all Giardia assemblages including A, B, and E may be due to an environment contaminated with animal and human feces in this village.

Cross-sectional studies of tuberculosis prevalence in Cambodia between 2002 and 2011.

OBJECTIVE: To measure trends in the pulmonary tuberculosis burden between 2002 and 2011 and to assess the impact of the DOTS (directly observed treatment, short-course) strategy in Cambodia. METHODS: Cambodia's first population-based nationwide tuberculosis survey, based on multistage cluster sampling, was conducted in 2002. The second tuberculosis survey, encompassing 62 clusters, followed in 2011. Participants aged 15 years or older were screened for active pulmonary tuberculosis with chest radiography and/or for tuberculosis symptoms. For diagnostic confirmation, sputum smear and culture were conducted on those whose screening results were positive. FINDINGS: Of the 40,423 eligible subjects, 37,417 (92.6%) participated in the survey; 103 smear-positive cases and 211 smear-negative, culture-positive cases were identified. The weighted prevalences of smear-positive tuberculosis and bacteriologically-positive tuberculosis were 271 (95% confidence interval, CI: 212-348) and 831 (95% CI: 707-977) per 100,000 population, respectively. Tuberculosis prevalence was higher in men than women and increased with age. A 38% decline in smear-positive tuberculosis (P = 0.0085) was observed with respect to the 2002 survey, after participants were matched by demographic and geographical characteristics. The prevalence of symptomatic, smear-positive tuberculosis decreased by 56% (P = 0.001), whereas the prevalence of asymptomatic, smear-positive tuberculosis decreased by only 7% (P = 0.7249). CONCLUSION: The tuberculosis burden in Cambodia has declined significantly, most probably because of the decentralization of DOTS to health centres. To further reduce the tuberculosis burden in Cambodia, tuberculosis control should be strengthened and should focus on identifying cases without symptoms and in the middle-aged and elderly population.

Optimization of the microscopic observation drug susceptibility assay for four first-line drugs using Mycobacterium tuberculosis reference strains and clinical isolates.

OBJECTIVE: The aim of this study is to determine the appropriate cut-off value and turnaround time of the microscopic observation drug susceptibility assay (MODS) for isoniazid (INH), rifampicin (RMP), streptomycin (STR), and ethambutol (EMB). DESIGN: A total of 39 Mycobacterium tuberculosis strains with confirmed drug susceptibility (reference strains) were tested with a range of drug concentrations to determine the optimal cut-off values for INH, RMP, STR, and EMB by MODS. Standard drug susceptibility testing (DST) results were evaluated relative to the Löwenstein-Jensen (L-J) proportion method. Following which, the performance of MODS was evaluated again using 36 sputum samples from patients with tuberculosis (TB) using the cut-off values determined in the aforementioned process. RESULTS: With 39 reference strains, DST identified the following cut-off values: 0.8µg/ml INH (sensitivity, 96.0%; specificity, 92.9%), 2.0µg/ml RMP (sensitivity, 100%; specificity, 95.5%), 4.0µg/ml STR (sensitivity, 90.5%; specificity, 93.8%), and 4.0µg/ml EMB (sensitivity, 100%; specificity, 91.7%). When these cut-off values were used to analyze the 36 clinical isolates, the sensitivity and specificity of MODS were 100% and 93.1% for INH, 100% and 93.8% for RMP, 87.5% and 96.4% for STR, and 100% and 88.2% for EMB, respectively. The turnaround time for these clinical specimens was 9.0days by MODS (95% CI: 5.3-12.7), compared with 11.7days (95% CI: 9.5-13.9) for smear negative specimens. CONCLUSION: Our study identified the optimal cut-off values of the four first-line drugs for MODS based on a wide concentration range. With the optimal cut-off values determined in this study, MODS showed high discriminatory efficiency for DST. This study also demonstrated that MODS is useful for rapid diagnosis of drug-resistant TB even for a smear negative specimen, despite the fact that it generally uses smear positive specimens as direct DST.

Comparative evaluation of three immunochromatographic identification tests for culture confirmation of Mycobacterium tuberculosis complex.

BACKGROUND: The rapid identification of acid-fast bacilli recovered from patient specimens as Mycobacterium tuberculosis complex (MTC) is critically important for accurate diagnosis and treatment. A thin-layer immunochromatographic (TLC) assay using anti-MPB64 or anti-MPT64 monoclonal antibodies was developed to discriminate between MTC and non-tuberculosis mycobacteria (NTM). Capilia TB-Neo, which is the improved version of Capilia TB, is recently developed and needs to be evaluated. METHODS: Capilia TB-Neo was evaluated by using reference strains including 96 Mycobacterium species (4 MTC and 92 NTM) and 3 other bacterial genera, and clinical isolates (500 MTC and 90 NTM isolates). M. tuberculosis isolates tested negative by Capilia TB-Neo were sequenced for mpt64 gene. RESULTS: Capilia TB-Neo showed 100% agreement to a subset of reference strains. Non-specific reaction to M. marinum was not observed. The sensitivity and specificity of Capilia TB-Neo to the clinical isolates were 99.4% (99.6% for M. tuberculosis, excluding M. bovis BCG) for clinical MTC isolates and 100% for NTM isolates tested, respectively. Two M. tuberculosis isolates tested negative by Capilia TB-Neo: one harbored a 63-bp deletion in the mpt64 gene and the other possessed a 3,659-bp deletion from Rv1977 to Rv1981c, a region including the entire mpt64 gene. CONCLUSIONS: Capilia TB-Neo is a simple, rapid and highly sensitive test for identifying MTC, and showed better specificity than Capilia TB. However, Capilia TB-Neo still showed false-negative results with mpt64 mutations. The limitation should be recognized for clinical use.

TRICORE, a novel bead-based specimen concentration method for the culturing of Mycobacterium tuberculosis.

Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently developed by Genetein Co., Ltd. We compared the efficacy of TRICORE and conventional centrifugation for concentrating M. tuberculosis in clinical sputum specimens by using liquid and solid culture systems. Among 90 pretreated clinical sputum specimens, 51 (57.3%) and 55 (61.8%) M. tuberculosis isolates were recovered by the MGIT culture system by using the centrifugation and TRICORE methods, respectively (chi-square test, p=0.5413). The detection time for the centrifugation method was 359.3±117.0 h, while that for the bead-based concentration method was 377.6±162.3 h (p=0.5637). However, the number of colonies recovered on solid media were significantly higher with the TRICORE method (p=0.003). In particular, among the smear-negative specimens, culture positivity of the TRICORE method was 39.6%, while that of the centrifugation method was 15.1%. The TRICORE bead-based concentration method was considered equivalent to centrifugation and enabled efficient collection of paucibacillary specimens in solution. Thus, the new noncentrifugation concentration method could yield more positive culture results.

[Evaluation of GenoType MTBDRplus for the detection of multi-drug-resistant Mycobacterium tuberculosis strains].

OBJECTIVE: To evaluate GenoType MTBDRplus (Hain Lifescience, Germany) for its capacity to detect the resistance of rifampicin (RFP) and isoniazid (INH). METHOD: A total of 44 confirmed multi-drug resistant (MDR) and 67 susceptible M. tuberculosis strains were tested for susceptibility to RFP and INH by GenoType MTBDR plus. The core 81bp region of the rpoB gene and the 322bp region of the katG gene and the inhA gene (248bp of which included the promoter and the ORF of the 379bp inhA) were directly sequenced for both MDR-TB and susceptible M. tuberculosis strains, and the mutations were confirmed. Susceptibility was tested by standard proportion method with 1% Ogawa medium. RESULTS: The sensitivities of GenoType MTBDRplus for RFP and INH resistance were 97.7% and 65.9%, respectively. The specificity for RFP and INH was 100%. The sensitivity of GenoType MTBDRplus was almost equivalent to the sequencing method for RFP, but that for INH was slightly inferior to the sequencing without significant difference. Geno Type MTBDRplus detected 97.7% of the mutations of rpoB compared with the direct sequencing. It also detected 24 katG MUT1 (S315T1) (54.5%) and 5 inhA MUT1 (C15T) mutations (11.4%), while the direct sequencing detected an additional 2 (4.5%) katG mutants. DISCUSSION: The accuracy of GenoType MTBDRplus for the detection of RFP resistance was confirmed to be comparable to that of DST using conventional culture-based methods, while it was less accurate for detection of INH resistance. GenoType MTBDRplus is useful for early diagnosis and infection control for MDR-TB because it has a short turnaround time of approximately 6 hours.

Improved polyacrylamide-based artificial sputum with formalin-fixed tubercle bacilli for training of tuberculosis microscopists.

Sputum smear microscopy is an easy, inexpensive, and rapid method for detecting tubercle bacilli when there are more than 10,000 bacilli/ml in the original sputum. Furthermore, because the microscopic method provides not only quantitative, but also qualitative information, such as the shape of bacilli, it has remained significant. We have previously developed and reported panel test slides made from polyacrylamide-based artificial sputum (PBAS) mixed with both cultured THP-1 cells and nonpathogenic mycobacteria. In this paper, we report an improved preparation method for PBAS for panel test slides that provides a simplified method and enhanced availability with high consistency in each grade and in which only negative PBAS is prepared from polyacrylamide and cultured THP-1 cells and mixed with graded formalin-fixed Mycobacterium tuberculosis solution (FFTBS) containing oral flora and Pseudomonas aeruginosa on the slides. In the smears prepared using this improved method, the numbers (average ± standard deviation [SD]) of acid-fast bacilli (AFB) in 300 fields (2- by 3-cm smear) in eight smears of each grade ranged from 5 to 9 (6.4 ± 1.4), from 59 to 88 (74.6 ± 10.0), from 503 to 912 (705.0 ± 145.7), and from 1,819 to 3,256 (2133.3 ± 478.0) in ±, +, ++, and +++ smears, respectively. In addition, this preparation method provided high similarity to the microscopic appearance of bacilli and background seen in the actual patient sputum, with high feasibility. These results revealed that our new PBAS had high authenticity in the appearance and consistency in each grade, which could make it valuable as a reliable artificial sputum for the training of microscopists.