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Vibrio cholerae can cause a range of symptoms, from severe diarrhea to asymptomatic infection. Previous studies using whole-genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous mutations in V. cholerae DNA repair genes in 5 out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome and in 1 of 3 patients with isolate genomes sequenced. Second, these mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, nonmutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCE Pathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease or asymptomatic infection. Here, we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

Background Although asymptomatic carriage of Streptococcus pneumoniae (Spn) is common, acquisition of the bacteria is the first step in disease pathogenesis. We examined the effect of introduction of the 7-valent pneumococcal vaccine on Spn carriage patterns in a cohort of Peruvian children. Methods We used data from a prospective cohort study that collected monthly nasopharyngeal samples from children under 3 years of age. Spn isolates were serotyped using Quellung reactions, and bacterial density was determined by quantitative polymerase chain reaction. Changes in Spn carriage patterns, including the rate of carriage and number and density of serotypes carried over time, were evaluated before (2009) and after widespread vaccination with PCV7 (2011). Using all pneumococcal detections from each child and year, we identified serotypes that were present both at first and last detection as “persisters” and serotypes that replaced a different earlier type and were detected last as “recolonizers.” Results Ninety-two percent (467/506) of children in 2009 and 89% (451/509) in 2011 carried Spn at least once. In 2009 and 2011, rates of carriage were 9.03 and 9.04 Spn detections per person-year, respectively. In 2009, 23F, a serotype included in PCV7, was the only type identified as a persister and 6A, 15B, and 19A were identified as recolonizer serotypes. In 2011, 6B and 7C were persister serotypes and 13 was a frequent recolonizer serotype. Conclusions Overall Spn carriage among children under 3 in Peru was similar before and after introduction of PCV7; however, serotype-specific rates and longitudinal carriage patterns have shifted.

PubMedID: 19034687 The prevalence of tinea capitis and the symptom-free colonisation of the scalp with dermatophytes were examined in 502 mentally retarded participants who attended day care centers in the Tarsus district, Mersin, Turkey. Between December 2006 and May 2007, a screening study was conducted in three centers on a total of 316 (62.9%) male and 186 (37.1%) female participants aged 12 ± 6.2 years. The examinations were carried out in parallel with the hairbrush, toothbrush, and cotton swab methods by inoculation onto Sabouraud glucose agar. No participant was diagnosed with tinea capitis; however, we detected three carriers, all of whom were boys aged 2-16 years. The total prevalence of carrier state was 0.6%. Of three boys, T. tonsurans was seen in two cases (66.7%), and in one case a zoophilic variant of T. mentagrophytes (33.3%) was isolated. The diagnosis was made via the hairbrush method in all three carriers. We also did a screening study on ten households of the three asymptomatic carriers. T. mentagrophytes also was isolated in a 5-year-old sister of the boy with T. mentagrophytes colonisation. All the carriers were followed-up without any antimycotic treatment. In two of the participants, the carrier state persisted at the 13th and 17th week follow-ups, and mycological clearance was documented at the 20th and 24th week for these individuals. The third case and the household's culture were found negative at the 7- and 12-week follow-ups. Despite poor hygienic conditions and the participants' difficulties in performing basic hygiene practices, asymptomatic carriage was found to be surprisingly less prevalent among the mentally retarded individuals. © 2008 Springer Science+Business Media B.V. Acknowledgments This study was supported by the Research Fund of Cukurova University (project no. TF2006BAP26).

BACKGROUND: Diagnosis of Pneumocystis jirovecii (PJ) pneumonia ordinarily requires invasive procedures that could be avoided by PCR methodologies, if these could be designed with adequate cut-off values for confounding background carriage.METHODS: We designed a novel quantitative real-time PCR assay to detect the mitochondrial large subunit rRNA gene of PJ in oral washes. To benchmark levels of PJ carriage versus infection, we tested asymptomatic immunosuppressed patients including Danish (n = 88) and West African HIV-infected (n = 142) patients, renal transplant recipients (n = 51), rheumatologic patients (n = 102), patients with inflammatory bowel diseases (n = 98), and healthy blood donors (controls, n = 50). The fungal burden in patients with PJ pneumonia (PCP, n = 7) was also investigated.RESULTS: Danish HIV-infected patients (with viremia/low CD4) and recent transplant recipients were at most risk of being carriers (prevalence of 23% and 16.7% respectively), whereas PJ was rarely detected among rheumatologic patients, patients with inflammatory bowel diseases, and untreated West African HIV patients. PJ was not detected among healthy controls. The fungal burden in patients with PCP fell rapidly on treatment.CONCLUSIONS: The quantitative PCR method described could conceivably discriminate between carriage and disease, given suitable threshold values for the former, and predict treatment efficacy by measures of the fungal burden in daily oral washes.

Thirty-year-old observations report frequent asymptomatic Clostridium difficile carriage among cystic fibrosis (CF) patients. In this case-control study, we found more carriers among CF patients than controls (47% versus 11%), but most strains carried by CF patients were non-toxigenic (77% versus 17%). Among CF patients, carriers were younger, with more severe pulmonary disease than non-carriers. Strains belonged to multiple PCR-ribotypes, suggesting that these CF patients did not acquire strains from each other.

SUMMARYLeishmaniaparasites can escape the immune response by invading cell types lacking leishmanicidal mechanisms. Silent persistence ofLeishmaniaparasites in the host organism is responsible for asymptomatic carriage and relapses after cured leishmaniasis. Here, we studied the interaction between Hepatic Stellate Cells (HSC) andLeishmania.An original model of human HSC in primary culture infected withL. donovaniwas developed. The presence of intracellular parasites was studied and quantified using optical and confocal microscopy. HSC characteristics were studied using microscopy, methylene blue assay, long-term cultures and qPCR. We showed for the first time that human HSC are permissive toL. donovaniinfection, with no modification of HSC survival, growth rate and proinflammatory and fibrogenic characteristics. Intracellular parasites did not replicate but HSC had no effect on their survival. Indeed, after a 40-day culture, infected HSC cultures transferred on NNN medium yielded new promastigotes that were able to proliferate and efficiently infect new cells. HSC are permissive toL. donovani, with neither parasite killing nor apparent cell damage. Thus, HSC could act as potent sanctuary cells forLeishmaniain the liver, which could partially explain parasite reactivation after an asymptomatic carriage or a cured visceral leishmaniasis.

Dissemination of Leishmania : a parasite sampled and picked up by the sandfly in the dermis of its vertebrate host. Study of an experimental model : Leishmania major and C57Bl/6 mouse. Parasites establish with their host a series of dynamic interactions, clinically visible or asymptomatic, on which depends their durability. Leishmania major is a protozoan parasite dependent on two hosts : (a) a hemophage and telmophage insect, the sandfly, and (b) a rodent. Following the inoculation, when and where do the Leishmania in their amastigote form migrate from the initial site ? In our laboratory, an infection model was developed based on current knowledge on hemophagia in telmophage sandflies. We prepared 1000 metacyclic promastigote Leishmania and inoculated them to C57Bl/6 mice via intradermal injections in the centre of the right pinna. This study, based on real-time/ quantitative PCR (qPCR) to quantify kinetoplastic DNA (kDNA), has shown that Leishmania were established in the centre of the inoculated pinna. On day 4 after the injection, we detected Leishmania kDNA in the periphery of that pinna and in the draining lymph node. The parasitic load grew regularly until day 28, then decreased between days 28 and 49. The maximum value recorded in the centre of the ear was 10,000 parasites on day 28, less in the periphery. The skin of the tail was the distant skin site where the detection of kinetoplastic kDNA was most consistent, starting on day 4. These results confirm the data on the kinetics of the parasitic load at the injection site. They also support the presence, within days after the inoculation, of a few parasites in asymptomatic distant cutaneous sites. The technique of qPCR to detect asymptomatic carriage has several applications both in Veterinary Medicine and in Public Health. The potential implications of these results on the parasite cycle are also emphasized., Qu’elles soient visibles cliniquement ou asymptomatiques, la pérennité des parasites est sous-tendue par les interactions dynamiques qu''ils établissent avec leurs hôtes. Leishmania major est un parasite protozoaire dépendant de deux autres organismes hôtes : (a) un insecte hématophage et telmophage, le phlébotome, (b) un rongeur. À partir de quand et où des leishmanies au stade amastigotes peuventelles émigrer du site initial où elles ont été inoculées ? Dans notre laboratoire, un modèle d’infection a été développé en tenant compte de ce qui est connu sur l''hématophagie par telmophagie des phlébotomes. 1000 leishmanies au stade promastigote métacyclique sont préparées et inoculées en face interne du pavillon auriculaire de souris C57Bl/ 6, par voie intradermique. Ce travail, fondé sur la quantification par PCR en temps réel (PCRq) de l’ADN kinétoplastique (ADNk), a permis de montrer que les leishmanies s’établissaient au centre de l’oreille (site de l’inoculation). Au quatrième jour après l''injection, nous avons détecté l’ADNk des leishmanies à la périphérie de cette même oreille et dans le ganglion drainant. La charge parasitaire dans le centre augmente régulièrement, jusqu’au jour 28, puis diminue entre les jours 28 et 49. Pour le centre de l’oreille, la valeur maximale a été ≈ 10 000 parasites au jour 28, elle est moindre pour le bord. La peau de la queue a été le site cutané distant où il a été possible le plus régulièrement de détecter de l’ADN kinétoplastique et ce, dès le quatrième jour. Outre la confirmation des données sur la cinétique de la charge parasitaire au site d’injection, nos résultats sont en faveur de l’existence, dans les jours qui suivent l’inoculation, de quelques parasites dans des sites cutanés asymptomatiques. Les applications de la PCRq dans le cadre du concept de portage asymptomatique concernent aussi bien la Médecine Vétérinaire que la Santé Publique. Les implications potentielles par rapport au cycle parasitaire sont soulignées., Marignac Geneviève, Lebastard Maï, Fall Ndeye Gamou, Nicolas Luc, Milon Geneviève. Exploration de la dissémination de Leishmania, un parasite délivré et prélevé par le phlébotome au niveau du derme de l'hôte Vertébré. In: Bulletin de l'Académie Vétérinaire de France tome 156 n°2, 2003. pp. 41-45.

SUMMARY Neisseria meningitidis is a cause of several life-threatening diseases and can be a normal commensal in the upper respiratory tract of healthy carriers. The carrier rate is not well established especially because there is no standard method for the isolation of N. meningitidis. Therefore, the aim of this study was to compare identification methods for the carrier state. Two swabs were collected from 190 volunteers: one was cultured and the other had DNA extracted directly from the sample. The Polymerase Chain Reaction (PCR) was performed to determine species and serogroups and compared the results between the methods. PCR for species determination used two pairs of primers and when there was only one amplicon, it was sequenced. The culture technique was positive in 23 (12.1%) subjects while the direct extraction method was positive in 132 (69.5%), p < 0.001. Among the 135 subjects with positive N. meningitides tests, 88 (65.2%) were serogroup C; 3 (2.2%) serogroup B; 5 (3.7%) were positive for both serogroup B and C, and 39 (28.9%) did not belong to any of the tested serogroups. In this study, PCR from DNA extracted directly from swabs identified more N. meningitidis asymptomatic carriers than the culture technique.

Background Group A Streptococcus (GAS; Streptococcus pyogenes) causes a range of mild to severe infections in humans. It can also colonize healthy persons asymptomatically. Therefore, it is important to study GAS carriage in healthy populations, as carriage of it might lead to subsequent disease manifestation, clonal spread in the community, and/or diversification of the organism. Throat swab culture is the gold standard method for GAS detection. Advanced culture-independent methods provide rapid and efficient detection of microorganisms directly from clinical samples. We investigated the presence of GAS in throat swab samples from healthy adults in Japan using culture-dependent and culture-independent methods. Results Two throat swab samples were collected from 148 healthy volunteers. One was cultured on selective medium, while total DNA extracted from the other was polymerase chain reaction (PCR) amplified with two GAS-specific primer pairs: one was a newly designed 16S rRNA-specific primer pair, the other a previously described V-Na+-ATPase primer pair. Although only 5 (3.4 %) of the 148 samples were GAS-positive by the culture-dependent method, 146 (98.6 %) were positive for the presence of GAS DNA by the culture-independent method. To obtain serotype information by emm typing, we performed nested PCR using newly designed emm primers. We detected the four different emm types in 25 (16.9 %) samples, and these differed from the common emm types associated with GAS associated diseases in Japan. The different emm types detected in the healthy volunteers indicate that the presence of unique emm types might be associated with GAS carriage. Conclusions Our results suggest that culture-independent methods should be considered for profiling GAS in the healthy hosts, with a view to obtaining better understanding of these organisms. The GAS-specific primers (16S rRNA and V-Na+-ATPase) used in this study can be used to estimate the maximum potential GAS carriage in people. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0858-5) contains supplementary material, which is available to authorized users.