Rattanaphone Phetsouvanh

Antimicrobial Drug Resistance of Salmonella enterica Serovar Typhi in Asia and Molecular Mechanism of Reduced Susceptibility to the Fluoroquinolones

ABSTRACT This study describes the pattern and extent of drug resistance in 1,774 strains of Salmonella enterica serovar Typhi isolated across Asia between 1993 and 2005 and characterizes the molecular mechanisms underlying the reduced susceptibilities to fluoroquinolones of these strains. For 1,393 serovar Typhi strains collected in southern Vietnam, the proportion of multidrug resistance has remained high since 1993 (50% in 2004) and there was a dramatic increase in nalidixic acid resistance between 1993 (4%) and 2005 (97%). In a cross-sectional sample of 381 serovar Typhi strains from 8 Asian countries, Bangladesh, China, India, Indonesia, Laos, Nepal, Pakistan, and central Vietnam, collected in 2002 to 2004, various rates of multidrug resistance (16 to 37%) and nalidixic acid resistance (5 to 51%) were found. The eight Asian countries involved in this study are home to approximately 80% of the worlds typhoid fever cases. These results document the scale of drug resistance across Asia. The Ser83→Phe substitution in GyrA was the predominant alteration in serovar Typhi strains from Vietnam (117/127 isolates; 92.1%). No mutations in gyrB, parC, or parE were detected in 55 of these strains. In vitro time-kill experiments showed a reduction in the efficacy of ofloxacin against strains harboring a single-amino-acid substitution at codon 83 or 87 of GyrA; this effect was more marked against a strain with a double substitution. The 8-methoxy fluoroquinolone gatifloxacin showed rapid killing of serovar Typhi harboring both the single- and double-amino-acid substitutions.

Rickettsial infections and fever, Vientiane, Laos.

Rickettsia spp. are an underrecognized cause of undifferentiated febrile illness.

Causes of non-malarial fever in Laos: a prospective study

Summary Background Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. Methods For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5–49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. Findings With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. Interpretation Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. Funding Wellcome Trust, WHO–Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention.

Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131

ABSTRACT Escherichia coli sequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n = 215) of sequenced ST131 E. coli isolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of a blaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration of blaCTX-M within subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, the blaCTX-M-14/14-like group. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli. These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages. IMPORTANCE Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specific E. coli lineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements. Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specific E. coli lineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.

Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium.

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an “inflammatory” monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.

Limited diagnostic capacities of two commercial assays for the detection of Leptospira immunoglobulin M antibodies in Laos.

ABSTRACT The diagnostic utility of immunochromatographic (Leptotek) and enzyme-linked immunosorbent assay (ELISA; Panbio) tests for the detection of Leptospira immunoglobulin M antibodies was assessed in febrile adults admitted in Vientiane, Laos. Both tests demonstrated poor diagnostic accuracy using admission serum (Leptotek sensitivity of 47.3% and specificity of 75.5%: ELISA sensitivity of 60.9% and specificity of 65.6%) compared to the Leptospira “gold standard” microscopic agglutination test.

Accuracy of Burkholderia pseudomallei Identification Using the API 20NE System and a Latex Agglutination Test

ABSTRACT In an evaluation of the API 20NE for the identification of Burkholderia spp., 792/800 (99%) Burkholderia pseudomallei and 17/19 (89%) B. cepacia isolates were correctly identified but 10 B. mallei and 98 B. thailandensis isolates were not correctly identified. A latex agglutination test was positive for 796/800 (99.5%) B. pseudomallei isolates and negative for 120 other oxidase-positive gram-negative bacilli.

Prospective Study To Determine Accuracy of Rapid Serological Assays for Diagnosis of Acute Dengue Virus Infection in Laos

ABSTRACT There is an urgent need for accurate and simple dengue virus infection diagnostic assays in limited-resource settings of dengue endemicity, to assist patient management. Using a panel of reference samples (S. D. Blacksell, P. N. Newton, D. Bell, J. Kelley, M. P. Mammen, D. W. Vaughn, V. Wuthiekanun, A. Sungkakum, A. Nisalak, and N. P. Day, Clin. Infect. Dis. 42:1127-1134, 2006), we recently evaluated eihgt commercially available immunochromatographic rapid diagnostic tests (RDTs) designed to detect dengue virus-specific immunoglobulin M (IgM) and/or IgG. We found that 6/8 RDTs had sensitivities of less than 50% (range, 6 to 65%), but specificities were generally high. Here, in conjuction with dengue virus serotyping by reverse transcriptase PCR and in the limited-resource setting of Laos, where dengue virus is endemic, we evaluated the same eight RDTs against a previously validated dengue IgM/IgG enzyme-linked immunosorbent assay for diagnosis of acute dengue virus infection. Paired serum samples were collected from 87 patients, of whom 38 had confirmed dengue virus infections (4 had primary infections, 33 had secondary infections, and 1 had an infection of indeterminate status). RDT sensitivity was low, with 7/8 RDTs having admission sample sensitivities of less than 20% (range, 4 to 26%). The majority (6/8) of the RDTs, demonstrated high specificity (>95%). Kappa statistic values ranged from 6 to 54% for the RDTs, demonstrating poor to moderate variation between three operators. No RDT adequately differentiated between primary and secondary dengue virus infections. The findings of this study suggest that currently available RDTs based on the detection of IgM antibodies for the diagnosis of acute dengue virus infections are unlikely to be useful for patient management.

Intrinsic fluoroquinolone resistance in Orientia tsutsugamushi

Scrub typhus is a public health concern for a population of over a billion humans, with an estimated incidence of one million cases/year in endemic areas. Although doxycycline remains the standard therapy, fluoroquinolones have been used successfully in a few patients. However, there is also clinical evidence that fluoroquinolones are ineffective in the treatment of scrub typhus. To clarify this matter, we determined the in vitro susceptibility of Orientia tsutsugamushi strain Kato to ciprofloxacin and ofloxacin and sequenced the quinolone resistance-determining region (QRDR) of the gyrA gene, the target of fluoroquinolones, of 18 fresh isolates from the Lao PDR. Orientia tsutsugamushi strain Kato was resistant to ciprofloxacin and ofloxacin in vitro (minimum inhibitory concentration=8 microg/mL). All sequences obtained, including those from the two available genomes of O. tsutsugamushi (strains Boryong and Ikeda), had a Ser83Leu mutation in their QRDR domain that is known to be associated with fluoroquinolone resistance. These findings re-emphasise the usefulness of in silico analysis for the prediction of antibiotic resistance and suggest that fluoroquinolones should not be used in the treatment of scrub typhus.

The infective causes of hepatitis and jaundice amongst hospitalised patients in Vientiane, Laos

Summary There is little information on the diverse infectious causes of jaundice and hepatitis in the Asiatic tropics. Serology (hepatitis A, B, C and E, leptospirosis, dengue, rickettsia), antigen tests (dengue), PCR assays (hepatitis A, C and E) and blood cultures (septicaemia) were performed on samples from 392 patients admitted with jaundice or raised transaminases (≥ × 3) to Mahosot Hospital, Vientiane, Laos over 3 years. Conservative definitions suggested diagnoses of dengue (8.4%), rickettsioses (7.3%), leptospirosis (6.8%), hepatitis B (4.9%), hepatitis C (4.9%), community-acquired septicaemia (3.3%) and hepatitis E (1.6%). Although anti-hepatitis A virus (HAV) IgM antibody results suggested that 35.8% of patients had acute HAV infections, anti-HAV IgG antibody avidity and HAV PCR suggested that 82% had polyclonal activation and not acute HAV infections. Scrub typhus, murine typhus or leptospirosis were present in 12.8% of patients and were associated with meningism and relatively low AST and ALT elevation. These patients would be expected to respond to empirical doxycycline therapy which, in the absence of virological diagnosis and treatment, may be an appropriate cost-effective intervention in Lao patients with jaundice/hepatitis.