Genotypic diversity of Streptococcus suis strains isolated from humans in Thailand.

The purpose of this study is to characterize Streptococcus suis isolates recovered from human infections regarding serotype distribution, genotypic profile, clinical manifestations, and epidemiology. A total of 668 S. suis isolates recovered from human infections in Thailand were characterized based on serotyping by multiplex PCR and co-agglutination, genotypic profiles by multilocus sequence typing, and PCR for virulence-associated genes, as well as review of medical records. Serotype 2 (94.6%) was predominant, followed by serotype 14 (4.5%), 24 (0.45%), 5 (0.3%), and 4 (0.15%). Multilocus sequence typing analyses revealed seven clonal complexes (CC): CC1 (56.43%), CC104 (31.74%), CC233/379 (5.4%), CC25 (4.5%), CC28 (0.9%), CC221/234 (0.6%), CC94 (0.15%), and two singletons. The CC1 group contained serotype 2 and 14 isolates, while CC25, 28, 104, and 233/379 consisted of serotype 2 isolates only. CC221/234 contained serotype 5 and 24 isolates, whereas the single serotype 4 isolate belonged to CC94. Two singletons contained serotype 5 (ST235) and 2 (ST236) isolates. Our data showed that ST1 isolates were more associated with meningitis than those of other STs (p < 0.001). The major route of infection was shown to be close contact with infected pigs or contaminated raw pork-derived products, including occupational exposure and recent consumption of raw pork products. This study revealed a relatively large number of CCs of S. suis causing human infection in Thailand. Among them, CC1 followed by CC104, with serotype 2 isolates, are predominant. Food safety campaigns and public health interventions would be important for controlling the S. suis infection in humans.

Impact of a Food Safety Campaign on Streptococcus suis Infection in Humans in Thailand.

AbstractStreptococcus suis is an important zoonotic pathogen in swine and humans that causes sepsis and meningitis. Our previous study in Thailand showed that the prevalence of S. suis infection in humans, especially in northern areas of Thailand, and the transmission of the pathogen occurred mainly through the consumption of traditional raw pork products. Considering the high incidence proportion and mortality rate of the disease as an important public health problem, we implemented a food safety campaign in the Phayao Province in northern Thailand in 2011. We evaluated the effects of a food safety campaign by comparing the sociodemographic, clinical, and bacteriological characteristics of cases before and after the campaign. The follow-up study showed a marked decrease of the incidence proportion in the first 2 years, indicating the effectiveness of the campaign. In the third year, however, the incidence proportion slightly increased again, indicating the existence of deep-rooted cultural behaviors and the necessity of continuous public health intervention. Furthermore, epidemiological analysis of the cases made it possible to estimate the infectivity of the pathogen via the oral route of infection. In the present study, we showed the effectiveness of the food safety campaign for controlling the S. suis infection, and we present a role model public health intervention for prevalent areas affected by S. suis infection in humans.

Adult pertussis is unrecognized public health problem in Thailand.

BACKGROUND: Although pertussis has been considered a disease of childhood, it is also recognized as an important respiratory tract infection in adolescents and adults. However, in countries with routine vaccination against pertussis with high coverage, pertussis is not usually taken into consideration for the etiology of prolonged cough in adults. Previous studies in a variety of populations in developed countries have documented that pertussis is quite common, ranging from 2.9 to 32% of adolescents and adults with prolonged cough. The anticipation and early recognition of this change in the epidemiology is important because the affected adolescents and adults act as reservoirs of the disease and source of infection to the vulnerable population of infants, for whom the disease can be life threatening. We conducted a prospective study to determine the prevalence of pertussis in Thai adults with prolonged cough. METHODS: Seventy-six adult patients with a cough lasting for more than 2 weeks (range, 14-180 days) were included in the present study. The data regarding medical history and physical examination were carefully analyzed. Nasopharyngeal swabs from all patients were obtained for the detection of deoxyribonucleic acid of Bordetella pertussis by the polymerase chain reaction (PCR) method. Paired serum samples were collected and tested for IgG antibody against pertussis toxin by using an ELISA method. RESULTS: Of 76 adult patients, 14 patients (18.4%) with the mean age of 59 (range, 28-85) years and the mean duration of cough of 34 (range, 14-120) days had laboratory evidence of acute pertussis infection. One patient was diagnosed by the PCR method, while the rest had serological diagnosis. Whooping cough is a significantly associated symptom of patients with chronic cough who had laboratory evidence of pertussis. (p < .05, odds ratio 3.75, 95% confidence interval: 1.00, 14.06) CONCLUSION: Pertussis is being increasingly recognized as a cause of prolonged, distressing cough among adults in Thailand. This result addresses the need of pertussis vaccination in Thai adults for preventing transmission to a high risk group such as newborn infants.

Contribution of the BacT/Alert MB Mycobacterium bottle to bloodstream infection surveillance in Thailand: added yield for Burkholderia pseudomallei.

Community-acquired bloodstream infections cause substantial morbidity and mortality worldwide, but microbiology capacity and surveillance limitations have challenged good descriptions of pathogen distribution in many regions, including Southeast Asia. Active surveillance for bloodstream infections has been conducted in two rural Thailand provinces for >7 years. Blood specimens were divided into two culture bottles, one optimized for aerobic growth (F bottle) and a second for enhanced growth of mycobacteria (MB bottle), and processed with the BactT/Alert 3D system. Because the routine use of MB culture bottles is resource intensive (expensive and requires prolonged incubation), we assessed the added yield of MB bottles by comparing the proportion of pathogens detected by MB versus that by F bottles from 2005 to 2012. Of 63,066 blood cultures, 7,296 (12%) were positive for at least one pathogen; the most common pathogens were Escherichia coli (28%), Burkholderia pseudomallei (11%), Klebsiella pneumoniae (9%), and Staphylococcus aureus (6%). Two bottles improved the yield overall, but the added yield attributable to the MB bottles was limited to a few pathogens. In addition to the detection of mycobacteria and some fungi, MB bottles improved the detection of B. pseudomallei (27% [MB] versus 8% [F]; P < 0.0001), with added benefit if therapy was initiated prior to the blood culture. The targeted use of MB bottles is warranted for patients at risk for mycobacterial and fungal infections and for infection with B. pseudomallei, a common cause of septicemia in Thailand.

Streptococcus suis infection in hospitalized patients, Nakhon Phanom Province, Thailand.

In Nakhon Phanom, Thailand, we identified 38 hospitalized patients with Streptococcus suis infection during 2006-2012. Deafness developed in 12 patients; none died. Thirty-five reported recent exposure to pigs/pork. Annual incidence was 0.1-2.2 cases/100,000 population (0.2-3.2 in persons ≥20 years of age). Clinicians should consider S. suis infection in areas where pig exposure is common.

The contribution of suilysin to the pathogenesis of Streptococcus suis meningitis.

BACKGROUND: Streptococcus suis is an emerging zoonotic pathogen, and causes sepsis and meningitis in humans. Although sequence type (ST) 1 and ST104 strains are capable of causing sepsis, ST1 strains commonly cause meningitis. In this study, we investigated the role of suilysin, a member of cholesterol-dependent cytolysins, in differential pathogenicity between ST1 and ST104 strains. METHODS: The levels of transcription and translation of the sly gene and messenger RNA of both ST strains were compared by means of quantitative polymerase chain reaction and Western blotting. Survival rates and bacterial densities in brain were compared between mice infected with wild-type and sly-knockout ST1 strain. ST104 infections with or without complementation of suilysin were also assessed. RESULTS: The amounts of suilysin produced by ST1 strains were much higher than those produced by ST104 strains. Lower production of suilysin by ST104 strains were attributed to the attenuated sly gene expression, which seemed to be associated with 2 nucleotide insertions in sly promoter region. Furthermore, suilysin contributed to the higher bacterial density and enhanced inflammation in brain and increased mortality. CONCLUSIONS: Our data may explain why ST1 strains, but not ST104 strains, commonly cause meningitis and also suggest the contribution of suilysin to the pathogenesis of meningitis in humans.

Incidence and etiology of acute lower respiratory tract infections in hospitalized children younger than 5 years in rural Thailand.

BACKGROUND: Pneumonia remains a leading cause of under-five morbidity and mortality globally. Comprehensive incidence, epidemiologic and etiologic data are needed to update prevention and control strategies. METHODS: We conducted active, population-based surveillance for hospitalized cases of acute lower respiratory tract infections (ALRI) among children <5 years of age in rural Thailand. ALRI cases were systematically sampled for an etiology study that tested nasopharyngeal specimens by polymerase chain reaction; children without ALRI were enrolled as controls from outpatient clinics. RESULTS: We identified 28,543 hospitalized ALRI cases from 2005 to 2010. Among the 49% with chest radiographs, 76% had findings consistent with pneumonia as identified by 2 study radiologists. The hospitalized ALRI incidence rate was 5772 per 100,000 child-years (95% confidence interval: 5707, 5837) and was higher in boys versus girls (incidence rate ratio 1.38, 95% confidence interval: 1.35-1.41) and in children 6-23 months of age versus other age groups (incidence rate ratio 1.76, 95% confidence interval: 1.69-1.84). Viruses most commonly detected in ALRI cases were respiratory syncytial virus (19.5%), rhinoviruses (18.7%), bocavirus (12.8%) and influenza viruses (8%). Compared with controls, ALRI cases were more likely to test positive for respiratory syncytial virus, influenza, adenovirus, human metapneumovirus and parainfluenza viruses 1 and 3 (P ≤ 0.01 for all). Bloodstream infections, most commonly Streptococcus pneumoniae and nontyphoidal Salmonella, accounted for 1.8% of cases. CONCLUSIONS: Our findings underscore the high burden of hospitalization for ALRI and the importance of viral pathogens among children in Thailand. Interventions targeting viral pathogens coupled with improved diagnostic approaches, especially for bacteria, are critical for better understanding of ALRI etiology, prevention and control.

Pneumococcal Bacteremia Requiring Hospitalization in Rural Thailand: An Update on Incidence, Clinical Characteristics, Serotype Distribution, and Antimicrobial Susceptibility, 2005-2010.

BACKGROUND: Streptococcus pneumoniae is an important cause of morbidity and mortality in Southeast Asia, but regional data is limited. Updated burden estimates are critical as pneumococcal conjugate vaccine (PCV) is highly effective, but not yet included in the Expanded Program on Immunization of Thailand or neighboring countries. METHODS: We implemented automated blood culture systems in two rural Thailand provinces as part of population-based surveillance for bacteremia. Blood cultures were collected from hospitalized patients as clinically indicated. RESULTS: From May 2005- March 2010, 196 cases of pneumococcal bacteremia were confirmed in hospitalized patients. Of these, 57% had clinical pneumonia, 20% required mechanical ventilation, and 23% (n = 46) died. Antibiotic use before blood culture was confirmed in 25% of those with blood culture. Annual incidence of hospitalized pneumococcal bacteremia was 3.6 per 100,000 person-years; rates were higher among children aged <5 years at 11.7 and adults ≥65 years at 14.2, and highest among infants <1 year at 33.8. The median monthly case count was higher during December-March compared to the rest of the year 6.0 vs. 1.0 (p<0.001). The most common serotypes were 23F (16%) and 14 (14%); 61% (74% in patients <5 years) were serotypes in the 10-valent PCV (PCV 10) and 82% (92% in <5 years) in PCV 13. All isolates were sensitive to penicillin, but non-susceptibility was high for co-trimoxazole (57%), erythromycin (30%), and clindamycin (20%). CONCLUSIONS: We demonstrated a high pneumococcal bacteremia burden, yet underestimated incidence because we captured only hospitalized cases, and because pre-culture antibiotics were frequently used. Our findings together with prior research indicate that PCV would likely have high serotype coverage in Thailand. These findings will complement ongoing cost effectiveness analyses and support vaccine policy evaluation in Thailand and the region.

Molecular characterization of antibiotic-resistant bacteria in contaminated chicken meat sold at supermarkets in Bangkok, Thailand.

We assessed contamination by antibiotic-resistant bacteria in chicken meat obtained from supermarkets in Bangkok, Thailand. The prevalence of Salmonella enterica and Escherichia coli was 18.7% (14/75) and 53% (106/200), respectively. Most probable number (MPN) analysis showed that 56.7% of the samples (34/60) were in violation of the limit of allowable coliform bacteria in chicken meat, for which the maximum is 46,000 MPN/g. Multidrug-resistant phenotypes of both S. enterica and E. coli were found. The presence of class 1 integrons was demonstrated by polymerase chain reaction (PCR) and dot-blot hybridization. PCR showed that class 1 integrons were present in 42.9% (6/14) and 37.7% (40/106) of S. enterica and E. coli isolates, respectively. Resistance genes identified in this study were aadA2, aadA4, aadA22, and aadA23 (for aminoglycoside resistance); dfrA5 (for trimethoprim resistance), and lnuF (for lincosamide resistance). Four S. enterica isolates underwent multilocus sequence typing and the results were sequence type (ST) 50, ST 96, ST 1543, and ST 1549, which matched well with strains from many countries and reflected an international spread. Our study revealed that class 1 integrons have spread into community sources and might play an important role in horizontal antibiotic resistance gene transfer.

Fifteen Streptococcus suis serotypes identified by multiplex PCR.

A multiplex PCR was developed to detect 15 serotypes of Streptococcus suis. This multiplex PCR was separated into two reaction sets. The first set identified nine serotypes (serotypes 1/2, 1, 2, 3, 4, 7, 9, 14 and 16) and the second set identified six serotypes (serotypes 5, 8, 10, 19, 23 and 25). This assay correctly detected serotypes 2, 5 and 14 in human isolates, and serotypes 1, 2, 1/2, 3, 4, 5, 7, 9, 14, 16 and 19 in pig isolates from Thailand. No cross-reaction was observed with other streptococcal species. This assay may be useful for the serotype surveillance of human and pig isolates of S. suis.

Genetic speciation of environmental Legionella isolates in Thailand.

Legionella-like organisms were isolated during 2003-2007 from various water resources by culturing on selective media of Wadowsky-Yee-Okuda agar. The 256 isolates were identified as belonging to the Legionella genus based on detection of 108 bp PCR product of the 5S rRNA gene, while the inclusion as Legionella pneumophila were confirmed by PCR detection of a specific mip gene region of 168 bp. The 50 isolates, identified as non-pneumophila, were then subjected to DNA tree analysis, based on mip gene of ~650 bp and rnpB genes product ranged from 304 to 354 bp. Phylogenetic tree was constructed to predict their species in relative to the available database. The isolates of which their speciation, based on those two genes were inconclusive, were then investigated for the almost full-length of 16S rRNA sequences. The isolates were assigned as 16 known Legionella species, and proposed seven novel species based on their unique 16S rRNA sequence.

Population-based study of Streptococcus suis infection in humans in Phayao Province in northern Thailand.

BACKGROUND: Streptococcus suis infection in humans has received increasing worldwide recognition. METHODS AND FINDINGS: A prospective study of S. suis infection in humans was conducted in Phayao Province in northern Thailand to determine the incidence and the risk behaviors of the disease in this region in 2010. Thirty-one cases were confirmed. The case fatality rate was 16.1%, and the estimated incidence rate was 6.2 per 100,000 in the general population. The peak incidence occurred in May. The median age of the patients was 53 years and 64.5% were men. Consumption of raw pork products was confirmed in 22 cases and the median incubation period (range) was 2 days (0-11) after consumption of raw pork products. Isolates from 31 patients were confirmed as serotype 2 in 23 patients (74.2%) and serotype 14 in eight patients (25.8%). The major sequence types (STs) were ST1 (n = 20) for serotype 2 and ST105 (n = 8) for serotype 14. The epidemiological analysis suggested three possible clusters, which included 17 cases. In the largest possible cluster of 10 cases in Chiang Kham and its neighboring districts in May, the source of infection in four cases was identified as a raw pork dish served at the same restaurant in this district. Microbiological analysis confirmed that three of four cases associated with consumption of raw pork at this restaurant were attributable to an identical strain of serotype 2 with ST1 and pulsotype A2. CONCLUSIONS: Our data suggest a high incidence rate of S. suis infection in the general population in Phayao Province in 2010 and confirm a cluster of three cases in 31 human cases. Food safety control should be strengthened especially for raw pork products in northern Thailand.

Streptococcus gallolyticus subspecies pasteurianus meningitis in an infant: a case report and literature review.

A case of 6-week-old male infant with meningitis and concurrent bacteremia caused by Streptococcus gallolyticus subspecies pasteurianus (Streptococcus bovis biotype 11.2) is presented. The isolates were susceptible to all beta-lactam antibiotics. Nevertheless, delayed defervescence and lack of satisfactory clinical improvement after treatment with multiple beta-lactam antibiotics prompted the combination therapy of vancomycin and penicillin G. The patient completed a 2-week course of antibiotics and recovered uneventfully.

Incidence of bacteremic melioidosis in eastern and northeastern Thailand.

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic in northeastern Thailand. Population-based disease burden estimates are lacking and limited data on melioidosis exist from other regions of the country. Using active, population-based surveillance, we measured the incidence of bacteremic melioidosis in the provinces of Sa Kaeo (eastern Thailand) and Nakhon Phanom (northeastern Thailand) during 2006-2008. The average annual incidence in Sa Kaeo and Nakhon Phanom per 100,000 persons was 4.9 (95% confidence interval [CI] = 3.9-6.1) and 14.9 (95% CI = 13.3-16.6). The respective population mortality rates were 1.9 (95% CI = 1.3-2.8) and 4.4 (95% CI = 3.6-5.3) per 100,000. The case-fatality proportion was 36% among those with known outcome. Our findings document a high incidence and case fatality proportion of bacteremic melioidosis in Thailand, including a region not traditionally considered highly endemic, and have potential implications for clinical management and health policy.

Genotypic profile of Streptococcus suis serotype 2 and clinical features of infection in humans, Thailand.

To examine associations between clinical features of Streptococcus suis serotype 2 infections in humans in Thailand and genotypic profiles of isolates, we conducted a retrospective study during 2006-2008. Of 165 patients for whom bacterial cultures of blood, cerebrospinal fluid, or both were positive for S. suis serotype 2, the major multilocus sequence types (STs) found were ST1 (62.4%) and ST104 (25.5%); the latter is unique to Thailand. Clinical features were examined for 158 patients. Infections were sporadic; case-fatality rate for adults was 9.5%, primarily in northern Thailand. Disease incidence peaked during the rainy season. Disease was classified as meningitis (58.9%) or nonmeningitis (41.1%, and included sepsis [35.4%] and others [5.7%]). Although ST1 strains were significantly associated with the meningitis category (p<0.0001), ST104 strains were significantly associated with the nonmeningitis category (p<0.0001). The ST1 and ST104 strains are capable of causing sepsis, but only the ST1 strains commonly cause meningitis.

Antibiotic use in Thailand: quantifying impact on blood culture yield and estimates of pneumococcal bacteremia incidence.

No studies have quantified the impact of pre-culture antibiotic use on the recovery of individual blood-borne pathogens or on population-level incidence estimates for Streptococcus pneumoniae. We conducted bloodstream infection surveillance in Thailand during November 2005-June 2008. Pre-culture antibiotic use was assessed by reported use and by serum antimicrobial activity. Of 35,639 patient blood cultures, 27% had reported pre-culture antibiotic use and 24% (of 24,538 tested) had serum antimicrobial activity. Pathogen isolation was half as common in patients with versus without antibiotic use; S. pneumoniae isolation was 4- to 9-fold less common (0.09% versus 0.37% by reported antibiotic use; 0.05% versus 0.45% by serum antimicrobial activity, P < 0.01). Pre-culture antibiotic use by serum antimicrobial activity reduced pneumococcal bacteremia incidence by 32% overall and 39% in children < 5 years of age. Our findings highlight the limitations of culture-based detection methods to estimate invasive pneumococcal disease incidence in settings where pre-culture antibiotic use is common.

Impact of pandemic influenza (H1N1) virus-associated community-acquired pneumonia among adults in a tertiary hospital in Thailand.

In July 2009, a pandemic influenza (H1N1) (pdm H1N1) virus epidemic emerged rapidly in Phitsanulok, Thailand. Adult cases of community-acquired pneumonia (CAP) were prospectively examined for pdm H1N1 virus infections by real-time PCR in a tertiary hospital in Phitsanulok from July to November 2009. Twenty-four cases of pdm H1N1 virus-associated CAP were confirmed, and their clinical features including bacterial infection, severity of disease, course of admission, treatment, and outcome were investigated. The median age of these cases was 39.5 years. Most cases appeared to be primary viral pneumonia, but only one case was positive for a urinary pneumococcal antigen. The median time from the onset of illness to admission was 4 days. All 24 patients received oseltamivir after admission. Twelve (50.0%) were defined as having severe CAP and 9 (37.5%) were diagnosed with acute respiratory distress syndrome (ARDS). During the study period, pdm H1N1 virus infections frequently caused severe CAP among young adults because of the delayed initiation of antiviral therapy. Of the 9 ARDS patients, 3 died of ventilator-associated pneumonia caused by multidrug-resistant Acinetobacter baumannii. Implementation of infection control targeting this pathogen is required in tertiary hospitals in Thailand.

Development of triplex SYBR green real-time PCR for detecting Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. without extraction of DNA.

Although Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. are prevalent causes of community-acquired pneumonia, rapid and sensitive diagnosis is difficult. Real-time PCR provides rapid and sensitive diagnosis, however, DNA extraction is still required, which is time-consuming, costly and includes a risk of contamination. Therefore, we aimed to develop triplex real-time PCR without DNA extraction. AmpDirect(R) Plus which inhibits PCR inhibitors was used as the PCR buffer. Melting temperatures of the PCR products for the three bacteria were analyzed by SYBR green triplex real-time PCR and were found to be significantly different. Detection limits of bacteria cells diluted in nasopharyngeal aspirates (NPAs) were comparable with the detection limits of previously reported real-time PCR. Our PCR without DNA extraction and probe real-time PCR with DNA extraction showed identical results for the detection of the three bacteria from 38 respiratory specimens (sputum, endotracheal aspirates, and NPAs) collected from patients with pneumonia. No cross-reaction with other bacteria was observed. Our triplex real-time PCR successfully detected and differentiated the three bacteria. Although further field tests are required, our assay is a promising method for the rapid and cost-effective detection of the three bacteria.

Invasive pneumococcal disease in Phramongkutklao Hospital 2004-2008: clinical data, serotype distribution and antimicrobial resistance patterns.

OBJECTIVE: To describe the clinical course, serotype distribution and antimicrobial resistance patterns of invasive pneumococcal disease (IPD) cases in a public hospital. MATERIAL AND METHOD: Retrospective review of IPD cases occurring from January 2004 through December 2008 was performed. Antibiotic susceptibility testing and serotyping were performed for available isolates. RESULTS: Fifty one IPD cases occurred during the study period, of which 47 had medical records available for review. The majority of cases occurred among children under 5 years of age (23.4%) and adults over 60 years of age (36.1%). Underlying diseases were identified in 72.3% of patients. Fifty-three percent of cases were associated with pneumonia, while 17% had meningitis, and 15% had isolated bacteremia. Serotype could be determined for 15 (31.9%) isolates, and 6B was most common. Based on current antibiotic susceptibility breakpoints for meningitis, 4 of the 7 available isolates from meningitis cases were penicillin resistant and one had reduced susceptibility to cefotaxime. Among non-meningitis isolates, 96.7% were penicillin susceptible and 3.3% had intermediate susceptibility to penicillin. Overall case fatality proportion was 19%. CONCLUSION: At this tertiary care hospital in Bangkok, IPD has disproportionately affected young children and the elderly. High rates of penicillin resistance among meningitis cases, the most severe form of IPD, underscore the need of appropriate treatment strategies and vaccine usage.