World Health Organization Reference Strains Research Articles

The Prevalence of Antibiotic Tolerance in Neisseria gonorrhoeae Varies by Anatomical Site.

Tolerance enables bacteria to survive intermittent antibiotic exposure without an increase in antimicrobial susceptibility. In this study, we investigated the presence of tolerance to three antimicrobials, ceftriaxone, azithromycin and ciprofloxacin, in clinical isolates and the WHO (World Health Organization) reference panel of Neisseria gonorrhoeae. We used the modified tolerance disk (TD test) to assess for tolerance to ceftriaxone, azithromycin and ciprofloxacin in 14 WHO reference strains and 62 N. gonorrhoeae clinical isolates-evenly divided between anorectal and urogenital infections. The isolates underwent a three-step incubation process wherein the isolates were exposed to an antibiotic disk for 20 h of incubation (Step I), followed by the replacement of the antibiotic disk with a nutrient disk for overnight incubation (Step II) and additional overnight incubation with extra nutrients (Step III). A total of 4 of the 62 clinical anorectal isolates and none of the urogenital isolates exhibited tolerance to azithromycin (p = 0.033). Tolerance to ceftriaxone and ciprofloxacin was observed in eight and four isolates, respectively, with no difference between infection sites. Tolerance was also detected in 8 (K, M, N, O, P, U, V, W) out of the 14 WHO reference strains, with varying patterns of tolerance to ceftriaxone (n = 8), ciprofloxacin (n = 2) and azithromycin (n = 1). This study identified ceftriaxone, azithromycin and ciprofloxacin tolerance in clinical and WHO reference N. gonorrhoeae isolates. Azithromycin tolerance was more common in anorectal than urogenital infections.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: An effective method for identification and phylogenetic analysis of Leishmania species

BackgroundMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and reproducible method that has been widely applied for the identification of bacteria and fungi. However, this technique has not yet been applied in clinical laboratories for parasitology, such as for the study of the protozoan Leishmania. MethodologyBy using MALDI-TOF MS, mass spectra database entries (MSPs) were created with 7 World Health Organization reference strains in order to establish a rapid method for Leishmania species identification. Furthermore, cluster analysis was performed with 18 Chinese Leishmania isolates. Principal findingsThe MSPs of Leishmania corresponded well with our past identification results, and the dendrogram analysis result was more or less similar to that of the phylogenetic analysis performed by multi-locus sequence typing. Conclusions/significanceMALDI-TOF MS is a promising method that offers both rapidity and efficiency for the identification and dendrogram analysis of Leishmania species.

Transmission Dynamics of the Rubella Virus Circulating in China During 2010-2019: 2 Lineage Switches Between Genotypes 1E and 2B.

To provide a better understanding of the progress on rubella control and elimination in China, a genetic analysis was conducted to examine the transmission pattern of the endemic rubella virus in China during 2010-2019. A total of 4895 strains were obtained from 29 of the 31 provinces in mainland China during 2010-2019. The genotyping regions of the strains were amplified, determined, and assembled. Genotyping analysis and lineage division were performed by comparisons with the World Health Organization reference strains and reported lineage reference strains, respectively. Further phylogenetic analyses were performed to compare the genetic relationship. During 2010-2019, the domestic lineage 1E-L1 and multiple imported lineages of rubella viruses including 2B-L1, 1E-L2, and 2B-L2c were identified. Further analysis of the circulation trend of the different lineages indicated that 2 switches occurred among the lineages. The first shift was from lineage 1E-L1 to 2B-L1, which occurred around 2015-2016, followed by the lowest rubella incidence in 2017. The second shift was from lineage 2B-L1 to 1E-L2 and 2B-L2c, which occurred around 2018-2019, coinciding with rubella resurgence and the subsequent nationwide epidemic during 2018-2019. Insufficient genomic information worldwide made it impossible to trace the origin of the imported viruses. China was moving toward rubella elimination, as evidenced by the fact that previous endemic lineages were not detected. However, rubella reemerged in 2018 2019 due to the newly imported rubella viruses. Therefore, to realize the rubella elimination goal, joint efforts are required for all countries worldwide.

Pathogenicity and transmissibility assessment of two strains of human influenza virus isolated in China in 2018.

ObjectiveInfluenza season occurs every year in China, but its presentation was unusual in the period from December 2017 to early 2018. During this period, influenza activity was increasing across the country and was much greater than during the same period in previous years, with great harm to people’s health.MethodsIn this study, we isolated two human influenza virus strains—A/Hebei/F076/2018(H1N1) and B/Hebei/16275B/2018—from patients with severe influenza in Hebei, China, during the flu season in January 2018, and explored their genetic characteristics, pathogenicity, and transmissibility.ResultsA/Hebei/F076/2018(H1N1) belongs to the human-like H1N1 influenza virus lineage, whereas B/Hebei/16275B/2018 belongs to the Victoria lineage and is closely related to the World Health Organization reference strain B/Brisbane/60/2008. Pathogenicity tests revealed that A/Hebei/F076/2018(H1N1) replicated much more strongly in mice, with mice exhibiting 40% mortality, whereas B/Hebei/16275B/2018 was not lethal. Both viruses could be transmitted through direct contact and by the aerosol route between guinea pigs, but the H1N1 strain exhibited higher airborne transmissibility.ConclusionsThese results may contribute to the monitoring of influenza mutation and the prevention of an influenza outbreak.

Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants.

The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally because of its reproductive health repercussions, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel (n = 15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles - established through phenotypic characterization and reference genome publication - and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 904 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635c, WHO_F_00745, WHO_F_01139, WHO_F_01144c, and WHO_F_01126, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates' defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics data bank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.

A World Health Organization Human Hepatitis E Virus Reference Strain Related to Similar Strains Isolated from Rabbits.

ABSTRACTWe report here the genome sequence of a hepatitis E virus (HEV) strain from a chronically infected immunodeficient patient. Full-length sequence analysis revealed a distinct HEV strain, of a tentative new subgenotype, clustering with viruses from rabbits. It is a World Health Organization reference strain for validation of nucleic acid testing.

Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing.

We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA.

Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

Prevalence and Antimicrobial Susceptibility Pattern ofNeisseria gonorrhoeaein a Tertiary care Hospital

According to World Health Organization (WHO) estimates, Gonorrhoea is one of the most common sexually transmitted infections (STIs) in developing countries. Control of gonococcal infection is becoming increasingly difficult due to emergence of resistant strains to a wide range of antibitiotics. Recent studies have revealed a high level of resistance against antibiotics such as, penicillins and quinolones in several countries, including India. There is irrational and injudicious use of antibacterial agents, especially in the developing countries like India and is expected to worsen unless urgent and appropriate steps are initiated. This study was done with an objective to find out the prevalence and antibiotic susceptibility pattern of Neisseria gonorrhoeae. This was a prospective study conducted in a tertiary care hospital in South India from June 2013 to May 2015. Samples were collected from patients attending gynecology and STD clinic. All patients in reproductive age group who attended gynecology out patient with suspected STIs were included in the study. Antimicrobial sensitivity test was performed by Kirby-Bauer disk diffusion method using CLSI guidelines. The susceptibility to the following antimicrobial agents were assessed by: Penicillin (10 IU), Tetracycline (30μg), Ciprofloxacin (5μg), Ceftriaxone (30μg), Cefixime (5μg), Cefoxitin (30μg), Cefotaxime (10μg), Nalidixic acid (30μg). The criteria used to select the antimicrobial agents tested were based on their availability. N. gonorrhoeae ATCC 49226 strain and WHO reference strains A, C, E, G, J, K-Q were used as controls. A total of 546 samples were analyzed, out of which 49 (9%) were positive for N. gonorrhoeae. Increase in N. gonorrhoeae isolates which are resistant to multiple antimicrobial agents is now a serious problem7. In the present study maximum resistance was seen with Penicillin and no resistance was seen with Ceftriaxone and Cefixime. There is a geographical difference in resistance pattern of N. gonorrhoeae, some centres have highlighted an alarming increase in the percentage of Penicillin and Quinolones over the years8, 9. Other studies from Nagpur (0%), Hyderabad (41.9%) and Kolkata (52.6%) have reported a lower percentage of Penicillin, findings similar to our study were reported from another WHO SEAR country, Thailand in 2013. Emergence of N. gonorrhoeae isolates with decreased susceptibility/resistance to ceftriaxone, as a consequence of excess utilisation of oral third-generation cephalosporins for other infectious conditions, may pose a serious threat in the management of gonorrhoea in countries like India. Antimicrobial surveillance should be done periodically to monitor the current susceptibility patterns in local hospitals. In most of the hospitals, sensitivity reports are not readily available, empirical therapy is often needed to. Regional antibiotic susceptibility studies will help in choosing an appropriate antibiotic for empirical therapy and reduce the mortality and morbidity.

Leishmania infantum and human visceral leishmaniasis, Argentina.

To the Editor: In Argentina, 14 autochthonous human cases of visceral leishmaniasis (VL) were reported during 1925–1989. These cases occurred in different localities in Salta, Jujuy, Santiago del Estero, and Chaco Provinces of northwestern Argentina (Figure A1), where cutaneous leishmaniasis (CL) caused principally by Leishmania (Viannia) braziliensis is endemic. It had been postulated that scattered/sporadic VL cases could be caused by visceralization of dermatrophic Leishmania spp. because of 1) absence of already recognized L. (Leishmania) infantum vector species; 2) geographic overlap with the region where CL is endemic; 3) simultaneous symptoms of CL; or 4) lack of detailed parasitic characterization at the molecular level for cases of suspected VL (1). However, during recent decades, urban outbreaks of VL have spread to southern regions of South America (Mato Grosso do Sul, Brazil, and Asuncion, Paraguay) near the northern border with Argentina. In May 2006, an autochthonous human case of VL was reported in Posadas (northeastern Argentina); it was associated with the canine visceral form of the disease. In addition, the presence of Lutzomyia longipalpis sandflies was also reported (2). Currently, 58 human VL cases have been reported in Posadas (3), and >7,000 infected dogs, Lu. longipalpis sandflies, and canine VL have been found 350 km south of Posadas (4). During 2007–2008, new VL cases in 4 children and 7 dogs were reported clustering in time and space in La Banda-Santiago del Estero in the dry Chaco region of Argentina. This focus showed a different pattern from that found in the only urban outbreak of VL reported (nearly the same number of cases in humans and dogs, and the suspected vector was Lu. migonei sandflies instead of Lu. longipalpis sandflies) (5). We report a case of autochthonous human VL in Salta Argentina that was caused by L. (L.) infantum. This parasite was characterized by cytochrome b (cytb) gene sequencing. Sequencing of this gene has been validated for precise characterization of Leishmania spp (6,7). On September 9, 2009, a 44-year-old man from Salta, Argentina (Figure A1), was admitted to the Infectious Disease Service at Hospital Senor del Milagro in Salta. The patient had fever, weight loss, dyspepsia, and splenomegaly that evolved over 3 weeks. Physical examination showed cutaneous and mucosal paleness. His general condition was feverish and rapidly deteriorating. Laboratory tests at the time of final diagnosis showed anemia, leukocytopenia, thrombocytopenia, and increased levels of lactate dehydrogenase. Results of urinalysis and coproculture were negative for parasites. Electrophoresis of serum proteins showed increased levels of gamma globulins. The differential diagnosis was negative for malaria, mycosis, autoimmune hepatitis, and lymphoma. A bone marrow smear showed abundant amastigotes by Giemsa staining (Figure, panel A). The patient was treated with liposomal amphotericin B, 3 mg/day for 7 days, and recovered (8). After a comprehensive interview, we verified that this patient had not been in the VL-endemic area in Argentina. However, he had worked (deforestation activities) during January–February 2009 on a farm in Finca Las Maravillas (22o3′29.30″S, 63o14′28.17″W), where he had been bitten by phlebotomines and acquired the disease. This farm was situated in the dry Chaco region near the border with Bolivia and Paraguay (zones with VL) (9), a region with intensive deforestation and agricultural activities. For species identification, DNA was extracted from a bone marrow aspirate and peripheral blood. We amplified by nested PCR and sequenced the cytb gene (Figure, panel B) (6). The aligned 817-bp sequence obtained showed 100% homology with the cytb gene of the MHOM/TN/80/IPT1 L. (L.) infantum World Health Organization reference strain (Tunisian strain) and 99.3% homology with the MHOM/BR/74/PP75 L. (L.) chagasi strain (Brazilian strain) (7). L. (L.) infantum was identified as the causative agent of this VL case in Salta, Argentina, where VL cases had not been seen for 50 years. Our findings indicate that this case was not caused by visceralization or a dermatropic Leishmania spp. We suggest that the scattered pattern of VL incidence in the dry Chaco region is caused by an enzootic cycle with accidental human transmission (5). There are no reports of Lu. longipalpis sandflies in the study area or surrounding areas (10). Nevertheless, studies on natural infections of vector sandflies and reservoir-host animals (especially dogs) are needed. Therefore, the search for naturally infected sandflies and reservoirs of this infection should be intensified. Epidemiologic surveys of dogs are needed to identify spread of VL foci in areas of deforestation. Deforestation could alter vector and reservoir range and parasite density in the enzootic cycle and increase human exposure to infected vectors. Figure Case of autochthonous human visceral leishmaniasis in a 44-year-old man, identified by parasitologic diagnosis and molecular detection of the causative species, Salta, Argentina. A) Leishmania amastigotes in a bone marrow smear. N, nucleus; K, kinetoplast; ...

Differences in the Antigenic Profile and Infectivity of Murine Macrophages of Leishmania (Viannia) Parasites

The antigenic profile and infectivity were compared between 3 recent Leishmania (Viannia) isolates from the Amazonian region (Instituto Nacional de Pesquisas da Amazonia [INPA] strains) and 3 World Health Organization (WHO) reference species (Leishmania guyanensis, Leishmania braziliensis, and Leishmania naiffi). Differences were observed in the peak and extent of promastigote growth. The WHO reference strains exhibited significantly higher exponential growth as promastigotes than INPA strains. In the immunoblot analyses, the INPA strains revealed several specific peptide fragments, as well as the greatest recognition frequencies by sera from Leishmania sp.-infected patients; among the latter, antigens derived from L. naiffi were the most frequently recognized. In vitro infection was carried out using mice peritoneal macrophages; all strains were able to enter the macrophages, but only L. amazonensis was able to reproduce. A striking observation was that L. naiffi exhibited the longest survival time inside the macrophages. Our data strongly suggest the application of recently isolated parasites as sources of antigen for diagnosis procedures. Moreover, L. naiffi species possesses several characteristics relevant for its use as a source of novel antigens to be explored in the design of diagnostic tools and vaccines.

Molecular characterization of Leishamania isolates from China by inter-simple sequence repeat polymerase chain reaction

Leishmania has distinct epidemiological and biological characteristics and causes a variety of clinical symptoms. To understand the genetic diversity and the phylogenetic relationships among Leishmania isolates from China, 29 Leishmania isolates from different geographic origins, vectors, and hosts were analyzed using 21 inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) primers. A total of 864 polymorphic bands were obtained. According to the results of the neighbor-joining phylogenetic tree and principal component analysis, the 29 isolates studied clustered into six groups. Isolates of Leishmania donovani complex from China share the highest similarity with the reference strain of L. donovani (DD8). This study helps to elucidate the genetic relationship among Leishmania isolates from China and similarities between Chinese isolates and World Health Organization reference strains. Furthermore, ISSR-PCR could also be a quick, simple, and reliable method for Leishmania species identification.

A Novel Tetravalent Dengue Vaccine Is Well Tolerated and Immunogenic against All 4 Serotypes inFlavivirus‐Naive Adults

BACKGROUND. Sanofi Pasteur has developed a tetravalent dengue vaccine (TDV) against the world's most common arbovirus infection. METHODS. We assessed the safety and immunogenicity of the TDV in healthy adults randomized into 2 groups. Group 1 received 3 TDV injections at months 0, 4, and 12-15; group 2 received saline placebo at month 0 and then 2 TDV injections at months 4 and 12-15. Adverse events were recorded, and biological parameters and viremia levels were measured. Neutralizing antibodies against 4 World Health Organization (WHO) reference strains were measured before and after vaccinations. RESULTS. A total of 33 participants were enrolled in each group. Demographic characteristics were comparable. No vaccine-related serious adverse event was reported. The most common systemic reactions were headache, malaise, and myalgia. Low viremia levels were detected, mainly of serotype 4. Immune response increased with successive vaccine doses. All participants seroconverted against all 4 serotypes after receiving 3 doses at 0, 4, and 12-15-months, and almost all seroconverted after 2 doses given 8-11 months apart. CONCLUSIONS. Sanofi Pasteur's TDV was well tolerated and induced full seroconversion against all WHO reference strain serotypes after 3 doses.

Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes

Emergence and spread of antimicrobial resistance (AMR) in Neisseria gonorrhoeae remain a major global problem and expanded, but valid, AMR surveillance is crucial for public health purposes. The World Health Organization (WHO) Collaborating Centre in Sydney, Australia, continually evaluates N. gonorrhoeae strains used in quality control and assurance aspects of the national, WHO regional and international programmes for AMR surveillance it conducts. Here we phenotypically and genetically characterized the 2008 WHO N. gonorrhoeae reference panel, widely used under existing WHO AMR surveillance protocols. The eight N. gonorrhoeae WHO reference strains were phenotypically characterized by antibiogram, auxotype, serovar and prolyliminopeptidase screening; and genetically with regard to resistance plasmid types, polymorphisms in divergent genetic resistance-mediating loci (n = 9), porB sequencing and N. gonorrhoeae multi-antigen sequence typing. The 2008 WHO reference strains represented all the important susceptible and resistant phenotypes, including corresponding resistance genotypes, and the range of resistances currently seen for relevant antimicrobials. Several pertinent additional phenotypic and genotypic markers, for example, epidemiological markers, were also determined. The 2008 WHO N. gonorrhoeae reference strain panel was extensively characterized, which is crucial for the expansion of gonococcal AMR surveillance nationally and internationally. The panel is available through WHO sources for quality assurance and quality control aspects of current phenotypic testing protocols, to allow valid comparison of AMR data derived by divergent methods, and also for the control of present and future molecular assays for AMR detection. Additional WHO reference strains can be included as required by the emergence of additional resistant phenotypes and/or genotypes.

Antimicrobial Susceptibility Profile of Resistance Phenotypes of Neisseria gonorrheae in India

Regular monitoring of antimicrobial resistance of Neisseria gonorrheae needs to be established in the country. The aim of the study was to determine the trend of resistance phenotypes of N. gonorrheae in India, and to assess the susceptibility profile of resistance phenotypes. Antimicrobial susceptibilities of N. gonorrheae isolates to penicillin, tetracycline, ceftriaxone, ciprofloxacin, and spectinomycin were determined by in terms of minimum inhibitory concentrations using Etest. World Health Organization reference strains were used as control strains. Phenotypic characterization of N. gonorrheae isolates was carried out on the basis of plasmid and chromosomally mediated resistance to penicillin and tetracycline. Of 382 isolates, 172 (45.0%) belonged to 6 resistance phenotype i.e., penicillinase-producing N. gonorrheae (PPNG) (16.5%), tetracycline resistant N. gonorrheae (TRNG) (8.9%), PPNG-TRNG (4.7%), chromosomally mediated tetracycline resistant N. gonorrheae (11.2%), and chromosomally mediated penicillin resistant N. gonorrheae (3.7%). Chromosomally mediated penicillin and tetracycline resistant N. gonorrheae isolates were not detected. Chromosomally mediated penicillin resistant N. gonorrheae and chromosomally mediated tetracycline resistant N. gonorrheae decreased from 2002 to 2006 whereas TRNG strains increased during the study period. Ciprofloxacin resistant and ceftriaxone less sensitive strains were more frequent among the resistance phenotypes. The high rates of plasmid and chromosomally mediated resistance to penicillin, tetracycline along with high rates of resistance to ciprofloxacin among all the resistance phenotypes underlines the necessity for continuous surveillance of antimicrobial resistance to help in controlling the spread of gonorrhoea.

Leishmaniasis Recidivans in a Palestinian Bedouin Child

In February 2005, a 12-year-old Palestinian Bedouin child with a small lesion on his upper cheek just below the right eye was referred to Leishmania Research Unit medical laboratory in Jericho for diagnosis of suspected cutaneous leishmaniasis. He lived in a rocky desert area by Wadi-Qelt, approximately 4 km from Jericho City center and attended the school in the adjacent Aqabat Jabr refugee camp, which borders Jericho City. He remained confined to this area for the few months before the lesion's appearance. The lesion was less than 1 cm in diameter, was bacterially clean, and had a small encrusted crater in its center. The lesion was 2 months old when first examined. There were no indications of previous cutaneous leishmaniasis, but other family members described having been exposed to cutaneous leishmaniasis. The family's residential area was rocky and cavernous with a continuously flowing stream that provided Jericho with drinking and irrigation water. There were irrigated banana plantations nearby. Extracellular amastigotes were seen in tissue smears stained with Giemsa stain (Figure 1). These parasites were identified as Leishmania tropica by DNA analysis. This was done by amplifying the internal transcribed spacer-1 of the ribosomal RNA genes, using a Gene Amp PCR-system 9700 (Applied Biosystems, Foster City, CA) followed by restriction fragment length polymorphism.1,2 The DNAs of leishmanial species reference strains were included for comparison (Figure 2). The patient was treated with sodium stibogluconate injected intralesionally at 10 mg/kg/d every other day for 3 weeks, then twice a week for 2 months, and once a week thereafter for 3 months. The lesion seemed to re-epithelialize during the 3 months from February to early May. The patient was lost to follow-up until November 2005, when he returned with a small, red, smooth macule. This enlarged, and papules typical of Leishmaniasis recidivans appeared at its margins. Microscopic examination and DNA analysis were repeated and again showed the presence of L tropica. The patient was treated with intralesional sodium stibogluconate for another 5 months. The patient returned a third time in April 2006, 14 months after initial presentation, with a more severe and destructive nodular condition (Figure 3). Microscopic examination and DNA analysis were repeated, during which 2 tubes of rabbit blood-agar semisolid medium were inoculated with tissue aspirate to isolate the parasite. This time, no amastigotes were seen in the stained smears, and the internal transcribed spacer-1-PCR results were negative, but culture medium in 1 tube grew promastigotes. The lesion remained active throughout further treatment. The Bedouin family said they would apply a traditional therapy. Figure 1. Giemsa-stained smear showing extracellular amastigotes (original magnification ×1000). Download figure to PowerPoint Figure 2. Restriction fragment length polymorphism analysis of the amplified internal transcribed spacer-1-Gene Amp PCR products (Applied Biosystems, Foster City, CA) following digestion with HaeIII: left to right, the World Health Organization reference strains of Leishmania infantum (MHOM/TN/1980/IPT1), Leishmania tropica (MHOM/AZ/1974/SAF-K27), and Leishmania major (MHOM/TM/1973/5ASKH); leishmanial DNA from the patient's samples taken in February 2005; and leishmanial DNA following the relapse in November 2005. Download figure to PowerPoint Figure 3. The 12-year-old Palestinian boy with Leishmaniasis recidivans, showing small papules at the edge of the central lesion after several months of almost complete cure. After 5 months of antimony treatment following relapse, promastigotes were isolated, while the microscopic examination and internal transcribed spacer-1-Gene Amp PCR (Applied Biosystems, Foster City, CA) results were negative. Download figure to PowerPoint

Molecular epidemiology of measles virus in Japan.

Measles virus has been classified into 22 genotypes. The present report examines the molecular epidemiology of measles virus in Japan from 1984 to 2002, and the epidemiological link between imported cases in several foreign countries and Japanese strains was elucidated from the literature. B95a or Vero cells was used to isolate the measles virus. The measles virus genome was amplified in the N and H genes by reverse transcriptase-polymerase chain reaction and were partially sequenced. Phylogenetic analysis of a partial sequence of the N gene, from position 1230 to 1685, of the recent measles strains was performed in comparison with the World Health Organization reference strains. There were large outbreaks of measles in Japan in 1984, 1987-1988, 1991-1993, and 2001-2002 and each outbreak was caused by a different genotype. Genotype C1 was an indigenous strain for a long period before 1985, while D3 was isolated in 1987-1988 and D5 in 1991-1993 outbreaks. In addition, the Chicago-type D3 caused sporadic regional outbreaks from 1998 to 1999. After 2000, H1 became the dominant circulating strain. It should be noted that the Japanese strains were detected as imported cases by epidemiological linkage in several countries. Among the recent circulating strain of measles virus in Japan the genotype H1 was dominant after 2000 and the Japanese strains D3, D5, and H1 were exported to several countries. It is recommended that Japan should adopt a more extensive and active vaccination strategy for measles elimination in line with other countries in the world.

Leishmania RNA viruses in Leishmania of the Viannia subgenus.

Karyotype analysis of 69 strains of Leishmania belonging to three species of the Viannia subgenus originating from the southeastern and southwestern regions of Colombia revealed approximately 5.3-kb RNAs in four strains of L. braziliensis and also in the World Health Organization reference strain L. guyanensis IWHI/BR/78/M5313. The RNA element in this reference strain and in L. braziliensis strains isolated from cutaneous and mucosal lesions of four patients hybridized with RNA probes prepared from cDNA of the RNA virus present in L. guyanensis strain CUMC-1-1A (LRV1-1). These strains also contained an 80-kD protein that reacted with polyclonal antibody prepared against a recombinant fragment of the coat (capsid) protein of LRV1-1. In addition, another Colombian strain of L. braziliensis was found to contain an approximately 3.5-kb RNA that did not hybridize with LRV1-1 probes. Contrasting with the strains containing the 5.3-kb RNA, a total lysate of this strain did not contain material reactive with antiserum to the capsid protein fragment. All Leishmania containing LRV1-related viruses identified to date have originated in the Amazon River basin. Karyotype analyses and biological characterization of 17 clones obtained from the highly metastatic L. guyanensis strain 5313 revealed retention of the approximately 5.3 kb RNA in all clones and no segregation of the virus with the metastatic trait. The restricted distribution of LRV1-related viruses among some strains of L. braziliensis and L. guyanensis circulating in the Amazon River basin makes these elements potential epidemiologic markers.

The characterization of Leishmania from patients with lymphadenopathy in Shiraz, Iran

Leishmania were cultured from enlarged lymph nodes of 6 of 18 patients with localized lymphadenitis and no other sign or symptom of leishmaniasis in Shiraz, Iran. The organisms failed to produce visible lesions when injected intradermally or intraperitoneally into Swiss outbred white or Balb/c mice. Golden hamsters similarly injected appeared well 16 weeks later, but histological examination of their spleens and livers revealed a few amastigotes in sections of the liver of one. Lectin agglutination profiles of promastigotes cultured from 2 of the patients were identical with those obtained with the World Health Organization reference strain of L. tropica (MHOM/SU/74/K27). Isoenzyme characterization of one of the isolates showed it to be L. tropica zymodeme lon18.

Reduced uptake and accumulation of norfloxacin in resistant strains of Neisseria gonorrhoeae isolated in Japan.

To investigate the alteration of cell permeability toward fluoroquinolones in Neisseria gonorrhoeae, which is a major quinolone-resistance mechanism along with the alteration of DNA gyrase in gram-negative bacteria. The prevalence of fluoroquinolone-resistant N gonorrhoeae strains is rapidly increasing in Japan. The uptake and accumulation of norfloxacin by gonococcal cells, including six clinical and five World Health Organization (WHO) reference strains, were measured. Of the six clinical strains, two were highly resistant to norfloxacin (MIC 8.0 and 4.0 micrograms/ml), two were moderately resistant (MIC 1.0 and 0.5 microgram/ml), and two were sensitive (MIC 0.063 and 0.004 microgram/ml). All five WHO reference strains were sensitive to norfloxacin (MIC < or = 0.001 to 0.063 microgram/ml). Mean initial norfloxacin uptake in the four resistant strains (104 ng/mg of dry cells) was significantly lower than that in the seven sensitive strains (158 ng/mg of dry cells) (p < 0.05). The mean uptake after 20 minutes was also significantly lower in the four resistant strains (130 ng/mg of dry cells) than in the seven sensitive strains (194 ng/mg of dry cells) (p < 0.05). However, there was no significant difference in mean norfloxacin accumulation after 20 minutes between the four resistant strains (26 ng/mg of dry cells) and the seven sensitive strains (36 ng/mg of dry cells). The accumulation of norfloxacin after 20 minutes was almost zero in two of the four resistant strains, while the remaining two strains accumulated norfloxacin as well as the sensitive strains. These findings suggest that alteration of bacterial cell permeability is a quinolone-resistance mechanism in N gonorrhoeae isolated in Japan, and that this bacteria may exhibit other mechanisms such as alteration of DNA gyrase.