Infect Immun. 2013 Jul;81(7):2426-36.

Regulation of Rab5 function during phagocytosis of live Pseudomonas aeruginosa in macrophages.

Mustafi S, Rivero N, Olson JC, Stahl PD, Barbieri MA.

Department of Biological Sciences, Florida International University, Miami, Florida, USA.



Pseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa. These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5.

PMID: 23630954

Fig 1. Pseudomonas aeruginosa uses Type III secretion mechanism and injects effector proteins in host cells. Exoenzyme S (EXOS) is a type III secretion effector protein which ADP-ribosylates small GTPases and modify their activity inside host cells. Rab5, a small GTPase protein is a key regulator of early phagocytosis. Rab5 cycles between GDP- bound inactive form and GTP-bound active form, which are regulated by several Rab5-GAPs (Guanine Activating Proteins) and Rab5-GEFs (Rab5-Guanine Exchange Factors). In this study it was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis, and that this occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on EXOS ADP-ribosyltransferase activity and more than one arginine residue in Rab5 are possible target for the ADP-ribosylation modification.

Vaccine 2013 Nov;31(48):5729-5735.

Intranasal immunization with a non-adjuvanted adhesive protein descended from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection in mice. 

Sasaki H, Ishikawa H, Kojima K, Itoh M, Matsumoto T, Itoh T, Hosomi O, Kawamoto E



Intranasal vaccination is one of the most effective means of protecting against invading and colonizing pathogens because the vaccine elicits a mucosal immune response. The exploitation of vaccine adjuvants and delivery systems for intranasal vaccines is an important way to evoke antigen immunogenicity and elicit a better immune response at the mucosal sites. In the present study, we assessed the potential of intranasal immunization using a non-adjuvanted bacterial adhesive protein toward the host organs. We evaluated intranasal immunization with modified recombinant PnxIIIA (MP3) from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection caused by P. pneumotropica, without using any adjuvants or delivery systems. The 100-kDa MP3 was confirmed to retain its immunogenicity and binding activity to collagen type I similar to the parent PnxIIIA. When MP3 was fused to green-fluorescent protein and inoculated into C57BL/6J mice intranasally, fluorescence intensity in the intranasal airway could be observed until 3 h after inoculation. Mice were intranasally immunized with MP3 at a maximum of 4 doses, with 7-day intervals. The antibody titer of serum IgG and IgA specific for MP3, as well as that of bronchoalveolar lavage fluid IgA, showed more than 9 (log₂) after 3 or 4 rounds of immunization. Experimentally infecting immunized mice with P. pneumotropica resulted in the inability to isolate the bacterium from the nasal cavity, trachea, conjunctiva, or cecum with more than 3 doses in the immunized mice. Although the detection in each organ seldom changed with less than 2 rounds of immunization, unlike that observed in the non-immunized mice, the detection remarkably decreased with 3 or more rounds of immunization. These results suggest that intranasal immunization with a non-adjuvanted adhesive protein could have preventive effects against opportunistic infection by P. pneumotropica.

PMID: 24091313



     Intranasal vaccination is one of effective methods to elicit mucosal and systemic immune response.  In particular, IgA is known to be the most abundantly synthesized isotype, and secretory IgA (sIgA) is also known to defend mucosal surface against invasive pathogens.  To induce mucosal immune response, design of vaccine antigen and adjuvant required for intranasal vaccination is important issues.

     The study by Sasaki et al. demonstrated the intranasal immunization using collagen-binding protein, termed as PnxIIIA derived from rodent pathogen P. pneumotropica (Pp) in mice.  Although putative virulence associations are involved, the PnxIIIA was modified to less cytotoxicity protein MP3 with retaining its immunogenicity and collagen-binding activity.  Multiple MP3 intranasal vaccinations successfully prevented mice against opportunistic infection by Pp (Figure).  This study did not focus on only prevention of Pp infection in mice but broad perspective of mucosal immunization strategy.  In brief, MP3 itself is considered to act as a delivery protein, and therefore intranasal vaccination of MP3 combined with the other pathogen’s antigen may help elicit the pathogen-specific IgA production.  Further, non-adjuvanted pathogens’ adhesive proteins themselves with ability to bind epithelial surface have possibility to induce protective sIgA production even in opportunistic infection.

Figure 1. Schematic reprensentation of non-adjuvanted MP3 intranasal immunization by Sasaki et al. P. pneumotropica PnxIIIA protein was modified to adequate vaccine immunogen, MP3 protein, with retaining immunogenicity and collagen-binding activity.  Non-adjuvanted MP3 was then immunized via intranasal route and was retained in nasal meatus for 3 or more hours after intranasal immunization.  MP3 retantion was observed GFP section in panel of murine nasal airway.  Multiple MP3 intranasal immunization prevented Pp infection.


Sasaki H, Ishikawa H, Kojima K, Itoh M, Matsumoto T, Itoh T, Hosomi O, Kawamoto E. Intranasal immunization with a non-adjuvanted adhesive protein descended from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection in mice. Vaccine. 2013 Nov;31(48):5729-5735.

PLoS One. 2013 Aug 30;8(8):e73465.

Development of the preterm gut microbiome in twins at risk of necrotising enterocolitis and sepsis.

Stewart CJ, Marrs EC, Nelson A, Lanyon C, Perry JD, Embleton ND, Cummings SP, Berrington JE.

Faculty of Health and Life Sciences, University of Northumbria, Newcastle upon Tyne, United Kingdom.



The preterm gut microbiome is a complex dynamic community influenced by genetic and environmental factors and is implicated in the pathogenesis of necrotising enterocolitis (NEC) and sepsis. We aimed to explore the longitudinal development of the gut microbiome in preterm twins to determine how shared environmental and genetic factors may influence temporal changes and compared this to the expressed breast milk (EBM) microbiome. Stool samples (n = 173) from 27 infants (12 twin pairs and 1 triplet set) and EBM (n = 18) from 4 mothers were collected longitudinally. All samples underwent PCR-DGGE (denaturing gradient gel electrophoresis) analysis and a selected subset underwent 454 pyrosequencing. Stool and EBM shared a core microbiome dominated by Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae. The gut microbiome showed greater similarity between siblings compared to unrelated individuals. Pyrosequencing revealed a reduction in diversity and increasing dominance of Escherichia sp. preceding NEC that was not observed in the healthy twin. Antibiotic treatment had a substantial effect on the gut microbiome, reducing Escherichia sp. and increasing other Enterobacteriaceae. This study demonstrates related preterm twins share similar gut microbiome development, even within the complex environment of neonatal intensive care. This is likely a result of shared genetic and immunomodulatory factors as well as exposure to the same maternal microbiome during birth, skin contact and exposure to EBM. Environmental factors including antibiotic exposure and feeding are additional significant determinants of community structure, regardless of host genetics.

PMID: 24023682



This paper explored the development of the gut microbiota in preterm infants from birth until discharge from a tertiary neonatal intensive care unit located in Newcastle upon Tyne, UK. This paper focused on twins and good longitudinal sampling facilitated an indepth analsysis of twins dicordant for necrotising enterocoloitis (NEC), that is one twin developed NEC (Patient 139) while the other did not (patient 140). NEC is the largest cause of mortality and morbidity in preterm infants in the developed world and despite research remains a challenging disease to prevent, diagnose and manage clinically. This paper offered important data using twins, providing the ideal diseased matched cohort, to show that Escherichia increased in abundance in the 10 days prior to the development of NEC. This genus has subsequently been categorised to species level as Escherichia coli, which is notably also present in healthy controls. Current work is currently focusing on strain level identification and the differences between the seemingly commensal E.coli and those involved in the pathology of NEC. It important that future studies go beyond sequence data to explore the functional roles of these organisms in disease causality.

Fig 1. Development of gut microbiota in twin pair 139/140 mapped to life events. P – Penicillin, G – Gentamicin, F- Fluctoxacillin, A – Amoxycillin, M – Metronidazole, s –Start of antibiotics, e – End of antibiotics, 72hr – full enteral feed (at least 150 ml/kg/day) sustained for 72 hours. a) Shannon Diversity indices (H’) of twin pair based on DGGE data. b) Turnover of the most prevalent bacterial OTUs throughout the first 36 days of life in twin 139 where antibiotics were prescribed for NEC. c) Turnover of most prevalent bacterial OTUs throughout the first 34 days of life in twin 140 where antibiotics were prescribed due to pyrexia (fever).

Infect Immun. 2014 Jan;82(1):243-52.

Characteristic age distribution of Plasmodium vivax infections after malaria elimination on Aneityum Island, Vanuatu.

Kaneko A, Chaves LF, Taleo G, Kalkoa M, Isozumi R, Wickremasinghe R, Perlmann H, Takeo S, Tsuboi T, Tachibana S, Kimura M, Björkman A, Troye-Blomberg M, Tanabe K, Drakeley C.

Island Malaria Group, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.



Resurgence is a major concern after malaria elimination. After the initiation of the elimination program on Aneityum Island in 1991, microscopy showed that Plasmodium falciparum disappeared immediately, whereas P. vivax disappeared from 1996 onward, until P. vivax cases were reported in January 2002. By conducting malariometric surveys of the entire population of Aneityum, we investigated the age distribution of individuals with parasites during this epidemic in the context of antimalarial antibody levels and parasite antigen diversity. In July 2002, P. vivax infections were detected by microscopy in 22/759 individuals: 20/298 born after the beginning of the elimination program in 1991, 2/126 born between 1982 and 1991, and none of 335 born before 1982. PCR increased the number of infections detected to 77, distributed among all age groups. Prevalences were 12.1%, 16.7%, and 6.0%, respectively (P < 0.001). In November, a similar age pattern was found, but with fewer infections: 6/746 and 39/741 individuals were found to be infected by microscopy and PCR, respectively. The frequencies of antibody responses to P. vivax were significantly higher in individuals born before 1991 than in younger age groups and were similar to those on Malakula Island, an area of endemicity. Remarkably low antigen diversity (h, 0.15) of P. vivax infections was observed on Aneityum compared with the other islands (h, 0.89 to 1.0). A P. vivax resurgence was observed among children and teenagers on Aneityum, an age distribution similar to those before elimination and on islands where P. vivax is endemic, suggesting that in the absence of significant exposure, immunity may persist, limiting infection levels in adults. The limited parasite gene pool on islands may contribute to this protection.

PMID: 24166950



Recently, the scaling up of malaria control efforts in endemic countries has shown some promising results. This has led to renewed interest in malaria elimination with 39 countries stating their commitment to achieve elimination. Since these countries are all positioned along the endemic margins, prevention of reinfection and resurgence is an integral component of any elimination campaign. In the Asia Pacific region, the unique challenge for elimination relates to the relatively high proportion of Plasmodium vivax infections. Islands provide natural ecological experiments with a great potential for intervention studies and have demonstrated some early successes of malaria elimination (5). Vanuatu consists of 68 islands in Southwest Pacific with a high linguistic diversity. Despite different waves of human colonization, unstable malaria transmission has continued probably since the first human settlement 4000 years ago. Aneityum, the southernmost island in Vanuatu, is located at the south-east edge of the malaria extension in the Pacific. To examine the feasibility of malaria elimination, an integrated control program, combining mass drug administration and vector control, was initiated on Aneityum in 1991. Eight years later, it was concluded that malaria can be eliminated from isolated islands if there is a high degree of community commitment. One major concern is the possible reintroduction of infection due to inter-island human movement. To our knowledge, Aneityum is the only island in recent times where malaria elimination has been successfully maintained for more than a decade. Thus, observations from Aneityum can offer important insights into concerns regarding loss of anti-malarial immunity following elimination and how this might impact disease burdens in potential resurgences. An epidemic of P. vivax on Aneityum in 2002 provided us with an opportunity to investigate the age patterns of newly detected infections in the context of population level antibody responses to P. vivax and parasite antigen diversity. Individuals born before elimination had considerably fewer episodes of parasitemia than those born after elimination. Our findings indicate that protective immunity against P. vivax infections persists for a long time, at least 10 years, after the initiation of malaria elimination and thus absence of exposure to recurrent infections.



We thank the families in Vanuatu who participated in the study; James Yaviong and Sam Yamar for field surveys; Margareta Hagstedt for ELISA tests; Naoko Sakihama for genetic analyses; Mayumi Fukui, Ikuko Kusuda, and Isao Kimata for PCR tests; Tomomi Kuwana for study administration. This work (PI: A. K.) was supported by Swedish Research Council grants (2008-3097 and 2009-3233), Japan Society for Promotion of Science Grants (24390141, 22406008, Asia-Africa Science Platforms), Health Labour Sciences Research Grant and the Global COE Program at Nagasaki University. C. D. received grant support from the Wellcome Trust (091924). L. F. C. is funded by Program for Nurturing Global Leaders in Tropical and Emerging Communicable Diseases at Nagasaki University. This work is dedicated to late Professor Peter Perlmann.



  1. Kaneko A, Taleo G, Kalkoa M, Yaviong J, Reeve PA, Ganczakowski M, Shirakawa C, Palmer K, Kobayakawa T, Björkman A. 1998. Malaria epidemiology, glucose 6-phosphate dehydrogenase deficiency and human settlement in the Vanuatu Archipelago. Acta Trop. 1998;70:285–302.
  2. Kaneko A, Taleo G, Kalkoa M, Yamar S, Kobayakawa T, Björkman A. Malaria eradication on islands. Lancet. 2000; 356:1560–1564.


Akira Kaneko, MD, PhD
Karolinska Institutet
Island Malaria Group
Department of Microbiology,
Tumor and Cell Biology (MTC)
Nobels vägen 16
SE-17177 Stockholm
Akira-Figure-1Figure 1. Age-specific prevalence profiles for P. vivax infections in July 2002 and IgG antibodies to P. vivax antigens in 1998 (bars) on residents of Aneityum island, where P. falciparum malaria transmission had been interrupted since 1991 and P. vivax since 1996. Red bars represent the numbers of P. vivax infections by both microscopy and PCR, and light blue bars those by only PCR. All microscope positives were PCR-positive. Dark blue bars represent seropositive rates for antibodies to P. vivax erythrocyte stage antigens and orange bars those for antibodies to P. vivax CSP recombinant proteins, either VK210 or VK247.


Figure 2. The Vanuatu archipelago: distribution of Pvmsp1 and Pvcsp haplotypes in P. vivax parasites on Aneityum island during the outbreak in 2002, a decade after beginning the malaria elimination program (n= 27 & 25 for Pvmsp1 & Pvcsp, respectively) and other islands with malaria transmission, Gaua (n= 33 & 20, in 1997), Santo (n= 34 & 32, in 1996, 1997, & 2001), Ambae (n= 22 & 11, in 2002), Malakula (n= 14 & 12, in 1998 & 2001), Pentecost (n= 20 & 14, in 1998 & 2000), and Tanna (n= 15 & 11, in 1999 & 2002). See Tables 1 & 2 for the detailed haplotype classifications for Pvmsp1 and Pvcsp, respectively. Parasite rates detected during the case-selection surveys on these islands are presented in the map. The lower inset map shows the location of Vanuatu in Oceania.

Akira-Figure-3Fig 3. Akira on Aneityum island, February 2014.

Med Chem Res.2014 March 02. DOI: 10.1007/s00044-014-0964-6

Recent progress and challenges in the computer-aided design of inhibitors for influenza A M2 channel proteins.

Linh Tran, Ly Le.

Ho Chi Minh Int Univ, Sch Biotechnol, Quarter 6,Linh Trung Ward, Thanh Pho Ho Chi Minh, Vietnam.



The M2 channel protein has become an attractive target for the design of new drugs against influenza because it plays a crucial role in the replication cycle of influenza A virus. Several adamantane-based drugs have recently been developed to inhibit the activity of the M2 channel and overcome the drug resistance issues observed in amantadine and rimantadine. Computer-aided drug design continues to play a critical role in the drug discovery process in terms of its contribution to the identification and development of new therapeutic agents. Scientists working in this field are currently facing significant challenges with regard to creating novel platforms capable of enhancing our understanding of these proteins, with computational techniques being used to search for new potential drugs against influenza. This review provides a summary of recent progress in drug discovery toward the development therapeutic agents targeting M2 channel proteins. It is hoped that this review will stimulate the development of new strategies for overcoming drug resistance problems and encourage the design of new and improved drugs against influenza A virus.



The importance of computer-aided drug design has grown significantly in terms of its contribution to the drug development process [1]. Numerous reports have demonstrated that theoretical computational studies, including molecular modeling, molecular docking, molecular dynamics simulations, phylogenetic analysis, quantum mechanical calculations, pharmacophore modeling, QSAR, and bioinformatics techniques can provide useful information for research in drug development [2-4]. Several computational studies have been conducted on the mechanisms associated with M2 channel proteins, and the results of these studies have provided valuable insights into the activity of the M2 channel proteins and enhanced efforts toward the targeting of M2 channel proteins through rational drug design.

Several studies and reviews have recently been reported pertaining to M2 channel proteins that have provided an in-depth understanding of its characteristic features, including its inhibition mechanisms and general structure. The M2 channel protein is activated by low pH and consists of three distinct segments, including: an extracellular N-terminal segment (residues 1–23), a transmembrane (TM) segment (residues 24–46) and an intracellular C-terminal segment (residues 47–97) [5]. The four TM helices create a channel where the His37 residue acts as a pH sensor, the Trp41 residue acts as proton gate, and the Asp44 residue acts as a channel lock [6].

Understanding the mechanisms involved in the inhibition of M2 channel proteins is essential for defining a basic research strategy and conducting a drug development program against this target. We believe that the functional binding site of M2 channel proteins has finally been identified. The amino acid residues inside the M2 channel proteins (i.e., the residues from V27 to G34) have been identified as being particularly important to favorable adamantane inhibition. These results also providing an understanding of why most of the residues inside the M2 proton channel are highly conserved.

The identification and characterization of correlated mutations within the M2 protein could provide an additional source of information with regard to the functional significance of specific residues and regions of the viral proton channel.

The anti-influenza drugs amantadine and rimantadine, which target the M2 channel protein of the influenza A virus, are no longer effective against this virus because of the spread of drug resistance as a consequence of five key mutation points including S31N, L26F, V27A, A30T, G34E, and L38F. The S31N is known to be the most dominant of these mutations, and is present in almost all of the circulating influenza A strain. Phylogenetic study also revealed that several specific positions, including 27, 28, 31, 36, 43, 50, 54, and 57, were involved in mutational correlations clusters [7]. Only a few of these mutational points have previously been described as being significant to the proton transfer mechanism. The roles played by the residues at positions 28, 36, 50, 54, and 57, however, still remain unknown.

We have reviewed several new strategies in computational drug design that used state-of-the-art techniques, as well as a review of recent progress in both computational and experimental studies aimed at identifying new lead compounds for the development of M2 channel inhibitors. The gap in our current understanding of the 3D structures of M2 channel proteins and their inhibition mechanisms has recently been established, which has led to an increase in the amount of research being conducted toward drug resistance and novel rational drug design.



This work was supported by Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) through grant numbers 106.01–2012.66. The computing resources and support provided by the Institute for Computational Science and Technology, Ho Chi Minh City, Vietnam is gratefully acknowledged.



1. Sukumar N, Das S (2011) Current trends in virtual high throughput screening using ligand-based and structure-based methods. Comb Chem High Throughput Screen 14(10):872–888

2. Tran L, Choi SB, Al-Najjar BO, Yusuf M, Wahab HA, Le L (2011) Discovery of potential M2 channel inhibitors based on the amantadine scaffold via virtual screening and pharmacophore modeling. Molecules 16(12):10227–10255. doi:10.3390/molecules 161210227

3. Tran N, Tran L, Le L (2013) Strategy in structure-based drug design for influenza A virus targeting M2 channel proteins. Med Chem Res 22(12):6078–6088. doi:10.1007/s00044-013-0599-z

4. Wang JF, Chou KC (2012) Recent advances in computational studies on influenza a virus M2 proton channel. Mini Rev Med Chem 12(10):971–978

5. Pielak RM, Chou JJ (2011) Influenza M2 proton channels. Biochim Biophys Acta 1808 2:522–529. doi:10.1016/j.bbamem.2010.04. 015

6. Pinto LH, Holsinger LJ, Lamb RA (1992) Influenza virus M2 protein has ion channel activity. Cell 69(3):517–528 0092-8674(92) 90452-I

7. Le L, Leluk J (2011) Study on phylogenetic relationships, variability, and correlated mutations in M2 proteins of influenza virus A.PLoS ONE 6(8):e22970. doi:10.1371/journal.pone.0022970

Fig 1. Model structure of M2 channel protein (A), amantadine (B), and rimantadine(C)

Eur J Gynaecol Oncol. 2013;34(3):218-21.

Bioethical issues on the role of contemporary gynecologists concerning HPV vaccination.

Koumousidis A., Sofoudis Ch., Paltoglou G., Iavazzo Ch., Kalampokas Th., Tzoumas N., Salakos N.

Division of Colposcopy and Gynecological Oncology, 2nd Department OB-GYN, Aretaieion Hospital, University of Athens


Debate is heating up whether or not to require girls to be vaccinated against HPV, a leading cause of cervical cancer. Prolepsis against this plague is mainly focused on early detection with Pap-test (screening) and recently, on administrating HPV-vaccines in youths.

Objective: To discuss the augmented role of contemporary gynecologist in this bioethical, for young population, field in order to contribute to a desired decrease of the malignancy.

Material and methods: We searched the web (data-warehouse: articles, forums, etc., data-mining: sequence analysis, classification) for HPV-vaccination’s and related bioethical issues, extracting useful information.

Results: HPV-vaccines have already caused debates on whether they must be mandatory (Texas 2007) and on whether they cause a pseudo-safeness mental state, making youths “forget” annually necessary Pap-tests or, worse, urging them in promiscuity and eventually, increasing the cervical cancer’s occurrence.

Conclusions: Greece, in order to apply appropriately the Constitutional Law 5§5 (: All persons have the right to the protection of their health…), needs to train contemporary gynecologists in adequate youth’s consulting and proper family approaching on HPV-vaccination issues. Enhancing gynecologist’s role, vaccination’s effectiveness (sensitivity, specificity) will be augmented and on the other hand, a Rule of Social Law will be established in our country.

PMID: 23967549



Debate is heating up whether or not to require girls to be vaccinated against HPV, a leading cause of cervical cancer (CC). Prolepsis against this plague is mainly focused on early detection with Pap-test (screening) and recently, on administrating HPV-vaccines in youths. Bioethics as a harmonic joint that connects sciences together (life sciences, biotechnology, medicine, politics, law, and philosophy), reveals us several different canals of interaction among them in a way that on the one hand, practicing medicine can promote our personal, social, political, and philosophical consciousness while on the other hand, applying values of the latter sectors in the medical field, indicators like sensitivity or specificity and concepts such as «precaution», «therapy» and «follow-up» can be improved. Objective: To discuss the augmented role of contemporary gynecologist in this bioethical, for young population, field in order to contribute to a desired decrease of the malignancy. Material and methods: We searched the web (data-warehouse: articles, forums, and blogs, and applied data-mining analysis for HPV-vaccination’s and related bioethical issues: Association Rules (method for discovering interesting relations between variables within large databases), Sequence Analysis (comparison of sequences in order to find similarities, identification of intrinsic sequence features or sequence differences), Classification (identifying the sub-population to which new observations belong), Clustering (assignment of a set of observations into sub-sets (clusters) so that observations in the same cluster are similar in some sense), Forecasting (process of making statements about events whose actual outcomes (typically) have not yet been observed). We studied 98 different websites and made many different tables in order Association Rules, Sequence Analysis and Clustering to be properly applied. Results: Several “human” conclusions were made studying this web-population that was chosen using criteria, such as the quality and traffic of each website, the impact factor of various medical web-magazines, the potential voting (if any) of the on-line (e-)statements. We evaluated the quality of the written e-speech (grammar, syntaxes, structure of arguments/thoughts, etiology, conclusions), the qualifications (job, student) of the person who had made the e-statement, the described facts, the experiences and the habits that might have been reported in the e-context, the medical terminology (if any), the general personal beliefs/ethos of the writer.

For example, one of the websites (a forum discussion) that we checked out was:, and here is some useful material that was used: “HPV vaccine for my 17 year old daughter??….. Should I give my daughter this vaccine? Not sexually active yet, but the crowd says give it now. What does the glp community say? “ It is obvious that the way/quality of writing in combination with the cited aspect reveals us a web-person of low education-level, who has at least one daughter without early onset of her sexual contacts (>16 years old), tending mostly to choose “optional” on the vaccination issue. Her inquiry had been made in a forum of dubious quality/provided ideas (personal and not adequately, in the scientific sense of the word, proved), showing us her prejudiced stance, confusion, low level of information and increased fear towards the proposed medical methods. Moreover, in the same website in the page, the 11th answer urges women not to be advised by doctors while replying on the question: “Just got a CERTIFIED LETTER stating that I am late for my pap smear.“

HPV-vaccines have already caused debates on whether they must be mandatory (Texas 2007) and on whether they can cause a pseudo-safeness mental state, making youths “forget” annually necessary Pap-tests or, worse, urging them in promiscuity and eventually, increasing the cervical cancer’s occurrence. Discussion-Conclusions: The answer whether HPV vaccination should be mandatory or optional depends strongly on the culture of the population that this dilemma is addressed to, which mainly is a matter of the general education that is widely provided and moreover, of the spirit concerning the quality of the established law system within a democratic society. However, Bioethics, as a third force that intervenes in the system «mandatory-optional» seems to provide us with the happy medium to this dispute by just accepting both opinions and by applying the one according to the underneath educational and cultural matrix on which each society has been developed. As it is widely accepted, the doctor has always to inform the patient with honesty and respect to the inalienable right to the self-determination before taking any indicated medical action for precautionary, therapeutic, and/or other medical reasons. Proper parent’s as well as children’s consultation on a regular basis (every 6-12 months) before their child’s first coitus, would offer quite safe education before, during and after the administration of the vaccine. The State must give funds to ameliorate the training of the new gynecologists: books, extra training semesters in the specialty, conferences, programmes. This bioethical solution seems to be the only way to CC cases’ decrease and thus, to the increase in the sensitivity of the HPV/CC ‘s ‘precaution’ at large, including that of HPV-vaccination. Greece, in order to apply appropriately the Constitutional Law 5§5 (: All persons have the right to the protection of their health…), needs to train contemporary gynecologists in adequate youth’s consulting and proper family approaching on HPV-vaccination issues. Enhancing gynecologist’s role, vaccination’s effectiveness (sensitivity, specificity) will be augmented and on the other hand, a Rule of Social Law will be established in our country.

PLoS One. 2014 Jan 8;9(1):e84239.

The temporal trend of influenza-associated morbidity and the impact of early appearance of antigenic drifted strains in a Southeast Asian country.

Lian IeB1, Wu HD2, Chang WT1, Chao DY3.


1 Graduate Institute of Statistics and Information Science, National Changhua University of Education, Changhua, Taiwan.

2 Department of Applied Math, National Chung-Hsing university, Taichung, Taiwan ; Institute of Statistics, National Chung-Hsing university, Taichung, Taiwan.

3 Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung-Hsing university, Taichung, Taiwan.



Globally, influenza infection is a major cause of morbidity and mortality in the elderly, who are suggested to be the major target group for trivalent influenza vaccine (TIV) vaccination by World Health Organization. In spite of an increasing trend in vaccine coverage rates in many countries, the effect of vaccination among the elderly in reducing hospitalization and mortality remains controversial. In this study, we conducted a historical cohort study to evaluate the temporal pattern of influenza-associated morbidity among persons older than 64 years over a decade. The temporal patterns of influenza-associated morbidity rates among the elderly older than 64 years indicated that Taiwan’s elderly P&I outpatient visits have been decreasing since the beginning of the 1999-2000 influenza season; however, hospitalization has been increasing despite significant increases in vaccine coverage. The propensity score logistic regression model was implemented to evaluate the source of bias and it was found that the TIV-receiving group had a higher propensity score than the non-receiving group (P<0.0001). In order to investigate the major factors affecting the temporal pattern of influenza-associated morbidity, we then used the propensity score as a summary confounder in a multivariate Poisson regression model based on the trimmed data. Our final models suggested that the factors affected the temporal pattern of morbidity differently. The variables including co-morbidity, vaccination rate, influenza virus type A and B isolation rate were associated with increased outpatient visits and hospitalization (p<0.05). In contrast, variables including high propensity score, increased 1°C in temperature, matching vaccine strains of type A/H1N1 and type B were associated with decreased outpatient visits and hospitalization (p<0.05). Finally, we assessed the impact of early appearance of antigenic-drifted strains and concluded that an excess influenza-associated morbidity substantial trends toward higher P&I hospitalization, but not outpatient visits, during the influenza season with early appearance of antigenic-drifted strains.

PMID: 24416205



Infection by influenza viruses is a major cause of morbidity and mortality among all age groups globally, particularly in the group of 65 years and older. The way that influenza viruses can persistently circulated among human population involves two mechanisms: antigenic drift or antigenic shift. Antigenic drift is a mechanism for variation in viruses that involves the accumulation of mutations within the genes that code for antibody-binding sites. It results in a new strain of virus particles which cannot be inhibited as effectively by the antibodies generated by either previous infection or vaccination targeted against previous strains, making it easier for the virus to spread throughout a partially immune population. Antigenic shift is the process by which two or more different strains of influenza viruses, combine to form a new subtype having a mixture of the surface antigens of the two or more original strains. The example of antigenic shift will be 2009 pandemic influenza virus or recent discovered H7N9 influenza virus. As of antigenic shift, which occurred historically every 10 years, antigenic drift occurs every 2-3 years and the vaccines currently used against seasonal influenza contain antigens against three influenza strains (A/H1N1, A/H3N2 and B), which are altered yearly to target the strains that are predicted to circulate in the upcoming season by WHO. However, in the region of Southeast Asia and Recent surveys of influenza viruses in Taiwan by hemagglutination (HA) sequence comparisons have indicated that a high rate of vaccine mismatch and found that epidemic strains in this region often became the vaccine strains 2–3 years later for some unknown reasons. Generally, antigenic alterations of local strains that make existing antibody levels in the population no longer protective are considered to be related to disease severity, increased medical utility and vaccine efficacy.

The most effective way to combat against influenza virus infection is through vaccination. The yearly influenza vaccination of at-risk individuals became common practice worldwide after the Second World War. After 2000, more than 40 developed or rapidly developing countries recommended vaccination to prevent influenza and its complications for “high risk” groups, such as the elderly (65 years or older), patients with chronic conditions, and institutionalized populations. Nevertheless, vaccination of the elderly remains controversial. In spite of an increasing trend in vaccine coverage rate, pneumonia and influenza-associated hospitalization and mortality among the elderly have continued to rise in Italy, the United States and South Korea.

Similar results were also found in our temporal trend analysis of influenza-associated hospitalization. During year 2000-2009, the influenza outpatient visit rate gradually decreased for all age groups; however, the hospitalization rate gradually increased for only the older age groups (Fig 1). During the same time period, the yearly vaccination rates among the elderly older than 64 years of age increased. Although we don’t know the vaccination rate for age under 65 years old as it is relatively difficult to obtain, the increase of hospitalization rate instead of outpatient visit rates could be due to the change of the medical behaviors as the physicians tend to admit the elder patients into hospitals because of the insurance program or the increasing frailty/comorbidity of the elderly group.

Assessment of the long-term influenza vaccine effectiveness, defined by medical uses including outpatient visits and hospitalization, is complicated by the degree of match between vaccine and virus, vaccine delivery policy, individual vaccination history, gradual decline in immune competence with age, antibody quality and quantity providing cross-reactivity, medical accessibility, co-morbidity among the elderly and personal behaviors. In Taiwan, several policies implemented here make it advantageous to assess the long-term influenza vaccine effectiveness in the elderly including (1) the high coverage rate (over 97% at the end of 2003) of National Health Insurance (NHI) program implemented in Taiwan since 1995; (2) the Taiwan Centers for Disease Control (Taiwan-CDC) has coordinated a laboratory-based influenza virological surveillance network (Lab-ISN) starting in 2000 for providing up-to-date information on viral characteristics and activities; (3) The Taiwan program of targeted free influenza vaccination for people with underlying medical disorders was implemented since 1996 and further expanded in 1998 to include all people older than 64 years. Vaccines are delivered in health care settings by nurses or physicians, or in community settings through public health departments.

Although the vaccine clinical trial studies are most powerful in evaluating efficacy or effectiveness through randomization, it is expensive, smaller population and complicated by the circulation of influenza viruses matched with the vaccine strains. Using NHI research database could avoid the above problems but careful adjusting confounding factors will be crucial since the comparison groups are not randomized. Therefore, when we analyzed further using the poisson regression model, a propensity score was calculated to understand the unknown source of bias between vaccinated and un-vaccinated groups among the elderly group. We extracted as many as possible variables from the NHI research database regarding medical use behavior, medical condition, residential places and determine the most appropriate variables as surrogates to differentiate two groups.

After we finish the poisson regression model and use the model to predict the temporal trend of influenza morbidity, including outpatient visits and hospitalization, the graph was plotted against the observed numbers from the database, we could actually observe an unusual discrepancy during each epidemic and it was correlated with the virus isolation data obtained through Lab-ISN (Fig 2). This gave us an idea to calculate the excess morbidity by subtracting predicted morbidity numbers from the observed ones and the excess morbidity correlated with the appearance of antigenic-shifted influenza viruses. We found that an excess influenza-associated morbidity substantial trends toward higher P&I hospitalization, but not outpatient visits, during the influenza season with early appearance of antigenic-drifted strains.

In summary, our study concluded that the early appearance of antigenic-drifted strains of influenza viruses was correlated with the increased risk of P&I-associated hospitalization among the elderly. The inclusion of local strains which appeared antigenic-drifted based on the surveillance system in the composition of TIV among the elderly warrants careful consideration particularly in East or Southeast Asian countries.

fig-1Fig 1. The influenza outpatient visit rate and the hospitalization rate During year 2000-2009

Fig 2. Excess peak of influenza cases during 2000-2009 in Taiwan

J Microbiol Methods. 2013 Sep;94(3):175-9.

Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h.

Swift BM, Denton EJ, Mahendran SA, Huxley JN, Rees CE.

School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK.



The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne’s disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne’s disease. © 2013. Published by Elsevier B.V.

KEYWORDS: Bacteriophage, FASTplaqueTB assay, FPTB, Johne’s disease, MP, Magnetic separation, Media Plus, PMMS, Paratuberculosis, Peptide mediated magnetic separation, Rapid detection

PMID: 23811207



The slow growth of some pathogenic mycobacteria makes detection by traditional culture extremely difficult. For instance, culture results for M bovis can take up to eight weeks. Similarly, Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease, can take up to 16 weeks to culture. The long incubation times and poor levels of sensitivity achieved when culturing mycobacteria from blood limits its diagnostic power.

Methods such as PCR, are often used to detect bacterial DNA as an alternative to culture. Blood assays based on PCR amplification of genomic signature sequences from Mycobacterium have been described, but a major drawback of these methods are that they do not differentiate between living and dead cells. It is vital that only viable cells are detected rather than residual DNA from cells that have been inactivated, either by the host immune system or by treatment. Unfortunately, PCR-based bovine TB detection methods have been found to be limited both by specificity and sensitivity [1]. Thus methods of detecting viable cells are needed, that are faster and more sensitive that the current tests available.

Bacteriophages are viruses that infect bacterial cells. They can either be lytic (infect and lyse their host) or lysogenic (can infect and integrate with their hosts genome; for a schematic see Fig. 1). Phage have a specific host range and will only replicate within a viable cell. The ability of bacteriophage to infect and replicate within the slow growing mycobacteria’s own generation time has been the basis of many phage based detection methods [2]. A commercial phage-based detection platform has been developed for the detection of M. tuberculosis in sputum samples as a diagnostic test in people [3]. As the lytic phage used in the assay can infect a wide range of mycobacteria, this has led to a range of assay formats that can report on the presence of other viable mycobacterial cells in a sample within two days [4-6]. Rather than waiting for the growth of the mycobacterial cells, the assay monitors the replication of the bacteriophage in a viable host cell. The identity of the cell detected is then confirmed by PCR (Fig. 2). This can be done as a species-specific test [6] or multiplex PCR assays have been developed that will simultaneously report on the presence of a range of different pathogenic mycobacteria (bovine TB and MAP) [4]. These phage-based assays are low cost and do not need investment in specialist equipment or expensive reagents, and results are available within 48 hours.

RapidMAP (PBD Biotech Ltd.) uses mycobacteriophage D29 that has a broad host range within the Mycobacterium genus, including the faster growing non-pathogens. The assay has been successfully used to detect viable MAP in the blood of cattle with Johne’s disease. The results show that it is able to reproducibly detect low numbers of viable cells with better sensitivity than direct PCR and it even detected bacteria in the early stages of infection in blood ELISA-negative animals. The use of the broad-host range phage in the RapidMAP test means that other pathogenic mycobacteria, such as M. bovis, can be detected by simply changing the endpoint PCR. Thus the developed blood test is applicable to MAP and bTB in a veterinary setting.



B.M.C.S was funded by a Lab21/University of Nottingham PhD studentship. E.J.D. and S.M. were supported a BBSRC and Wellcome Trust Summer Studentships, respectively.


1. Parra A, Garcia N, Garcia A, Lacombe A, Moreno F, et al. (2008) Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis. Veterinary Microbiology 127: 315-324.
2. Monk AB, Rees CD, Barrow P, Hagens S, Harper DR (2010) Bacteriophage applications: where are we now? Lett Appl Microbiol 51: 363-369.
3. Albert H, Heydenrych A, Brookes R, Mole RJ, Harley B, et al. (2002) Performance of a rapid phage-based test, FASTPlaqueTB, to diagnose pulmonary tuberculosis from sputum specimens in South Africa. Int J Tuberc Lung Dis 6: 529-537.
4. Stanley EC, Mole RJ, Smith RJ, Glenn SM, Barer MR, et al. (2007) Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours. Applied and Environmental Microbiology 73: 1851-1857.
5. Botsaris G, Slana I, Liapi M, Dodd C, Economides C, et al. (2010) Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese. International Journal of Food Microbiology 141: S87-S90.
6. Swift BM, Denton EJ, Mahendran SA, Huxley JN, Rees CE (2013) Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h. J Microbiol Methods 94: 175-179.


Ben Swift –

Cath Rees –


Fig 1. Bacteriophage life cycle

Fig 2. Schematic of the RapidMAP phage assay.

Int J Infect Dis. 2013 Nov;17(11):e1000-4.

Non-tuberculous mycobacterial disease is common in patients with non-cystic fibrosis bronchiectasis.

Mirsaeidi M, Hadid W, Ericsoussi B, Rodgers D, Sadikot RT.

Department of Veterans Affairs, Jesse Brown VA Hospital, Chicago, Illinois, USA; Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois at Chicago, 840 South Wood Street, M/C 719, Chicago, IL 60612, USA.



Background: Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms. Cystic fibrosis (CF) patients are susceptible to NTM, but data about NTM in patients with non-CF bronchiectasis are limited. Methods: We conducted a retrospective, descriptive study at the University of Illinois Medical Center. All patients diagnosed with bronchiectasis (code 494) using the International Classification of Diseases, ninth revision (ICD-9), between 1999 and 2006, were identified. Clinical data including lung function, radiology studies, and presence of NTM in sputum were abstracted for those who met the study criteria. Results: One hundred eighty-two patients were enrolled in the study. Patients were divided into two groups: bronchiectasis with NTM isolates (n = 68) and bronchiectasis without isolates (n =114), and compared for clinical characteristics and underlying diseases. Mycobacterium avium complex (MAC) was the most common isolate. Fifty-five patients (30%) met the American Thoracic Society criteria for diagnosis of NTM disease. Gram-negative rods were commonly co-isolated. The probability of NTM isolation was significantly higher in elderly female patients (p = 0.04). Moreover, the probability of NTM isolation was significantly higher in the female group with low body mass index (BMI) (p = 0.002). Conclusions: NTM infections are common in non-CF bronchiectasis. MAC is the most frequently isolated NTM in these patients. There is also great variability in age and sex characteristics for NTM in non-CF bronchiectasis patients. Female patients with a low BMI are a high risk group for NTM infection in non-CF bronchiectasis. Routine screening for NTM is strongly recommended in this patient population.

Published by Elsevier Ltd.

KEYWORDS: BMI, Bronchiectasis, NTM, Non-CF, Non-tuberculous mycobacterial diseases

PMID: 23683809



Bronchiectasis represents a significant disease entity that has been historically under-represented in medical literature (1). It is primarily a disease of the bronchi and bronchioles involving a vicious circle of infection and inflammation. Bronchiectasis in general can manifest in one of two forms: as a local or focal obstructive process of a segment or lobe of a lung or as a diffuse process involving most of the lungs (2). When extensive it has the potential to cause devastating illness by predisposing susceptible individuals to recurrent respiratory infections. While the lower respiratory tract is normally sterile, conditions such as bronchiectasis enable colonization of a variety of microbes.

Recently, the incidence of NTM infections has been increasingly reported both in the immunocompromised and immunocompetent population. NTM pulmonary infection associated with bronchiectasis is increasing worldwide (3). The numbers of patients who die from nontuberculosis mycobacteria are increasing in the US (4).

The diagnosis of NTM pulmonary infection is often delayed because symptoms are mild and excretion of NTM in sputum is intermittent with few colonies retrievable in culture. Many patients therefore require bronchoscopic examination or lung biopsy for diagnosis of NTM pulmonary disease (5). Hence, there is limited information about characteristics of dual diseases of NTM and bronchestasis, we therefore investigated a cohort of patients with adult-onset bronchiectasis to determine the prevalence of NTM in this group. Clinical indices were also compared with patients with bronchiectasis who did not grow NTM over that time, to determine whether there was any association with disease severity, antibiotic usage, radiographical changes or microbiology.

The study was performed at the University of Illinois at Chicago. A retrospective, chart review, was conducted after obtaining an approval from the Internal Review Board (IRB). All patients diagnosed with Bronchiectasis using an ICD code (494) were identified between 1999 and 2006. A list of 306 patients with an ICD code 494 was generated. Patients were included in the study if they were 18 years and older and had confirmed radiological changes suggestive of bronchiectsis (chest X-ray or CT scan of the chest) reported by radiologist which was independently evaluated by two clinicians (Figure 1). Patients with cystic fibrosis were excluded (n=6). Demographic, clinical and laboratory data were collected.

Categorical variables were described as counts and percentages or examined as predictors using odds ratio and were tested by the χ2 or, if applicable, exact tests. Univariate analysis was used to compare differences in demographic and clinical variables between bronchiectasis patient whom NTM isolated and non-NTM patients. Continuous variables were compared using Student t test for normally distributed variables and Wilcoxon rank-sum test for non-normally distributed variables.

In order to examine risk factors for NTM isolation, a stepwise logistic regression model was used. The outcome variable was isolation of NTM from respiratory secretions (sputum or BAL) and other variables listed in Table 1, which were statistically significant upon univariate analysis ( P <0.10) or considered clinically relevant.

The Kaplan–Meier estimate curve was used to determine the cumulative probability of NTM isolation for age and BMI in each gender; curves between the two groups were compared using the log-rank test. All data analyses were performed using SPSS software for Windows, version 17.0 (SPSS), two-tailed P-values were used and P values <0.05 were considered to be statistically significant.

A hundred and eighty two patients diagnosed with non-CF bronchiectasis were included. Table 1 shows the demographic, clinical and outcome characteristics of the study population. Mean age (SD) of patients with diagnosis of bronchiectasis was 64.5 (15.1) years. From the 182 patients who were identified with a diagnosis of bronchiectasis within this time frame, NTM was isolated from sputum or BAL in 68 (37%) patients. Mycobactrium avium complex (MAC) was isolated from 55 (81%), M.Chelonae 5 (7%), M. Kansassi 2 (3%), and other NTM were isolated from 6 (9%) patients. The diagnosis of bronchiectasis and isolation of NTM was significantly higher in female patients. This was further confirmed by logistic regression analysis (Table 2). Patients in whom NTM was isolated also gave a significant history of previous childhood infections (Table 2).

The mean (SD) of diagnosis delay per months was 44.95 (56.2). Gram-negative rods (entrobacteriace group and Pseudomona arouginosa bacilli) were isolated from 22 patients (14%) of whom 17 (77%) never been co-isolated NTM (p=0.031). 41 (23%) patients were hospitalized secondary to complications from bronchiectasis of whom 7 (10.3%) patients were those with NTM (p=0.002). Table 2 shows multivariate model, which accounted for the possible confounding effect of several factors, for patients with bronchiectasis with and without NTM.

The univariate analysis suggested that patient with bronchiectasis and NTM used clearance devices more frequently although there was no difference in the rate of usage of antibiotics for respiratory infections. Overall seven patients with bronchiectasis during this period underwent surgery, 2 of who had NTM isolated and 5 patients were from Non NTM group. 55 (30%) patients met the ATS criteria for the diagnosis of NTM disease.

The probability of NTM isolation was significantly higher in elderly female patients with non-CF bronchiectasis (p = 0.04), as shown in Figure 2. Moreover, The probability of NTM isolation was significantly higher in female group with low BMI (P=0.002), as shown in Figure 3.

In summary, this study shows that NTM is commonly isolated from patients with non-CF bronchietasis. The frequency of NTM in our bronchiectatic population was 37% of whom 30% met the ATS criteria for NTM disease definition. Additionally MAC appears to be the most common pulmonary NTM (88%) in this group. The current study also shows that elderly females and patients with low BMI are at a higher risk for acquiring NTM diseases. A better understanding of the role of NTM in the outcomes of patients with bronchiectasis may generate new therapeutic modalities for patients with severe disease. Furthermore, during the initial assessment of patients with bronchiectasis, physicians might need to consider sputum culture, and possibly bronchoscopy if lung imaging study is suggestive for NTM as a predictive factor for clinical outcomes.

Table 1 Demographic, clinical, and outcome characteristics for patients with bronchiectasis with and without NTM.

Legend for table 1: Data are no. (%) of patients, unless otherwise indicated.*Fisher test


Table 2. Logistic regression analysis results for patients with bronchiectasis with and without NTM.

Legend for table 2: Adjusted risk of NTM isolation in patients with Non-Cystic Fibrosis bronchiectasis by: Age> 65 years old, Male gender, Recurrent childhood pulmonary infections, History of previous IV-Antibiotic therapy, P > 0.05 by Hosmer and Lemeshow goodness-of-fit test.




1. Smith MP. Non-cystic fibrosis bronchiectasis. J R Coll Physicians Edinb 2011; 41: 132-139; quiz 139.
2. Barker AF. Bronchiectasis. N Engl J Med 2002; 346: 1383-1393.
3. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society was approved by the Board of Directors, March 1997. Medical Section of the American Lung Association. Am J Respir Crit Care Med 1997; 156: S1-25.
4. Mehdi Mirsaeidi RFM, Joe G.N. Garcia, Dean E. Schraufnagel. Nontuberculous Mycobacterial Disease Mortality in the United States, 1999–2010: A Population-Based Comparative Study. PloS one 2014.
5. Huang JH, Kao PN, Adi V, Ruoss SJ. Mycobacterium avium-intracellulare pulmonary infection in HIV-negative patients without preexisting lung disease: diagnostic and management limitations. Chest 1999; 115: 1033-1040.

Microsoft Word - Figure 1.docxFigure 1. The flowchart of study of Non-tuberculous mycobacterial disease in patients with Non-Cystic Fibrosis bronchiectasis

 Microsoft Word - Figure 2.docxFigure 2. The Age function stratified for sex and Non-tuberculous mycobacterial disease in patients with Non-Cystic Fibrosis bronchiectasis

Microsoft Word - Figure 3.docx Figure 3. The BMI function stratified for sex and Non-tuberculous mycobacterial disease in patients with Non-Cystic Fibrosis bronchiectasis

Braz J Infect Dis. 2013 Mar-Apr;17(2):131-6.

Distribution of serotypes and evaluation of antimicrobial susceptibility among human and bovine Streptococcus agalactiae strains isolated in Brazil between 1980 and 2006.

Pinto TC, Costa NS, Vianna Souza AR, Silva LG, Corrêa AB, Fernandes FG, Oliveira IC, Mattos MC, Rosado AS, Benchetrit LC.

Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.



Streptococcus agalactiae is a common agent of clinical and subclinical bovine mastitis and an important cause of human infections, mainly among pregnant women, neonates and nonpregnant adults with underlying diseases. The present study describes the genetic and phenotypic diversity among 392 S. agalactiae human and bovine strains isolated between 1980 and 2006 in Brazil. The most prevalent serotypes were Ia, II, III and V and all the strains were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampicin and tetracycline was observed. Among the erythromycin resistant strains, mefA/E, ermA and, mainly, ermB gene were detected, and a shift of prevalence from the macrolide resistance phenotype to the macrolide-lincosamide-streptogramin B resistance phenotype over the years was observed. The 23 macrolide-resistant strains showed 19 different pulsed-field gel electrophoresis profiles. Regarding macrolide resistance, a major concern in S. agalactiae epidemiology, the present study describes an increase in erythromycin resistance from the 80s to the 90s followed by a decrease in the 2000-2006 period. Also, the genetic heterogeneity described points out that erythromycin resistance in Brazil is rather due to horizontal gene transmission than to spreading of specific macrolide-resistant clones. Copyright © 2013 Elsevier Editora Ltda.

PMID: 23453948



Streptococcus agalactiae, also known as group B Streptococcus (GBS), was first identified as a major cause of bovine mastitis during the late 19th century. Only in the mid 20th century this microorganism was also recognized as a frequent cause of invasive diseases among human newborns, and the epidemiological linkage between such different hosts has been the target of exhaustive studies for many years.

GBS epidemiology is still largely unknown in Brazil, and therefore we aimed to gather valuable information on this subject, by analyzing serotype distribution and antimicrobial susceptibility profiles of a large and diverse collection of GBS isolates, representing different periods of time and different isolation sources, which also enabled a comparison between human and bovine isolates.

Our results showed that serotype distribution was mainly stable along time, highlighting the prevalence of serotypes Ia, II, III and V (Figure 1). However, differences on serotype distribution were seen between human and bovine isolates, with serotype III prevailing among the former and serotype V among the last.

The most striking difference between GBS recovered from different hosts regarded erythromycin and clindamycin resistance. While around 4% and 2% of human isolates were resistant to erythromycin and clindamycin respectively, those numbers were around 30% and 20% for bovine isolates. Moreover, while erythromycin resistance levels showed a decreasing tendency along time among human isolates, an increasing trend was observed among bovine strains (Figure 2).

The emergence of erythromycin resistance has been linked to the vertical dissemination of specific GBS clones in some places of the world. In the present study, a great genetic diversity was seen among erythromycin-resistant isolates (Figure 3), suggesting that lateral gene transfer of resistance determinants might also play an important role in our setting. Still, some major clusters were observed, including cluster A, which was the most predominant and has been circulating since 1990. It is also worthy to highlight that closely related and even indistinguishable erythromycin-resistant clones were isolated from both human and bovine sources.

Preventive measures against GBS neonatal infections are usually based on the use of antibiotics. Although GBS is still largely susceptible to the first-line therapy which is represented by the beta-lactams, increasing rates of resistance to the alternative drugs erythromycin and clindamycin are being detected among isolates recovered from human sources in the last decades. Nevertheless, according to our results, erythromycin resistance has not fully emerged among GBS isolates recovered from humans in Brazil. The observation that it has, however, considerably increased among those recovered from cattle, especially in the last years, raises the question of how long this scenario will last among human GBS strains.



  1. Keefe GP. Streptococcus agalactiae mastitis: a review. Can Vet. 1997;38:429-437.
  2. Schuchat A. Epidemiology of group B streptococcal disease in the United States: shifting paradigms. Clin Microbiol Rev. 1998;11:497-513.
  3. Dogan B, Schukken YH, Santisteban C, Boor KJ. Distribution of serotypes and antimicrobial resistance genes among Streptococcus agalactiae isolates from bovine and human hosts. J Clin Microbiol. 2005;43:5899-5906.
  4. Oliveira ICM, de Mattos MC, Pinto TCA, et al. Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B Streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern. Clin Microbiol Infect. 2006;12:887-893.
  5. Gherardi G, Imperi M, Baldassarri L, et al. Molecular epidemiology and distribution of serotypes, surface proteins, and antibiotic resistance among group B streptococci in Italy. J Clin Microbiol. 2007;45:2909-2916.
  6. Brzychczy-Włoch M, Gosiewski T, Bodaszewska M, et al. Genetic characterization and diversity of Streptococcus agalactiae isolates with macrolide resistance. J Med Microbiol. 2010;59:780-786.
  7. Centers for Disease Control and Prevention. Prevention of Perinatal Group B Streptococcal Disease. Revised Guidelines from CDC. MMWR 59. 2010.

Fig-1-Pinto-et-al-2013Figure 1. Distribution of serotypes among Streptococcus agalactiae strains according to the period of isolation. Isolates from human and bovine sources were included, and nontypeable isolates were excluded.

Fig-2-Pinto-et-al-2013Figure 2. Distribution of erythromycin-resistant Streptococcus agalactiae (group B Streptococcus; GBS) isolates according to the period of isolation. An increasing trend of erythromycin-resistance occurrence was observed when all GBS isolates (in red) and those from bovine sources (in green) were considered, while human GBS strains (in blue) showed a decreasing tendency.


Figure 3. Genetic diversity among erythromycin-resistant Streptococcus agalactiae isolates by Pulsed-field gel electrophoresis (PFGE). Five major clusters were observed, three of which comprised both human and bovine isolates. Indistinguishable PFGE profiles were observed for both human and bovine isolates belonging to cluster E. cMLSB: constitutive macrolide, lincosamide and streptogramin B-type resistance; M: macrolide resistance; NT: nontypeable.

Clin Ther. 2013 Jul;35(7):904-14.

A novel method to value real options in health care: the case of a multicohort human papillomavirus vaccination strategy.

Favato G, Baio G, Capone A, Marcellusi A, Saverio Mennini F.

Institute of Leadership and Management in Health, Kingston University, London, United Kingdom.




A large body of economic evaluations has already confirmed the cost-effectiveness of different human papillomavirus (HPV) vaccination strategies. Standard analyses might not capture the full economic value of novel vaccination programmes, since the relative simplicity of the cost-effectiveness fails to take account of the value of active management. Management decisions can be seen as a real options, a term used to refer to the application of option pricing theory to the valuation of investments in non-financial assets where much of the value is attributable to flexibility and learning over time. Real option valuation is treating the different types of managerial flexibility as options and valuing them with option pricing models.


This study was aimed to determine the real option value of four HPV vaccination strategies – targeting girls aged 12, 15, 18 and 25 – in comparison with the outcomes of a Bayesian cost-effectiveness analysis.


This paper applies the recently developed pay-off method which derives the real option value from the pay-off distribution of the project’s NPV, which is treated as a fuzzy set. The possibilistic rather than probabilistic distribution of outcomes and the recognition of the economic value of the active role of management differentiate the pay-off pricing method for real options from the cost-effectiveness paradigm.


Costs per quality-adjusted life-year (QALY) gained appeared to be related to the number of cohorts targeted: a single cohort of girls aged 12 (€10,955; 95 CI: €1,021, €28,212) showed the lowest cost among the four alternative strategies evaluated. The real option valuation challenged the cost-effectiveness dominance of a single cohort of 12-year-old girls. The simultaneous vaccination of two cohorts of girls aged 12 and 15 yielded a real option value (€17,723) equivalent to that attributed to a single cohort of 12-year-old girls (€17,460).


A national vaccination programme targeting two cohorts should be the preferred method of implementation when the impact on access, coverage and, ultimately, time to prevention of HPV-induced diseases is taken into account. The use of both cost-effectiveness analysis and real option valuation could together improve the quality of policy-making under uncertain conditions, given the acknowledgement of the value of management in the implementation of healthcare programmes. Copyright © 2013 Elsevier HS Journals, Inc.

KEYWORDS: human papillomavirus, multicohort, real option, vaccination, valuation

PMID: 23806328



This paper presented a new method (pay-off method) to value real options in healthcare, which is intuitive to understand and less mathematically challenging than any previous real option pricing formula.

The main intuition behind the pay-off method is that management plays an active role in determining the economic value of the chosen project over its entire life. Like cost-effectiveness analysis, the pay-off method involves projecting future streams of costs and economic benefits, but its paradigm assumes that managers can influence the outcome by interventive actions that add value over time. By taking into account only the positive side of the distribution in the real option value, the pay-off method explicitly recognises the ability of managers to interrupt the intervention as soon as its net present value becomes negative, in order to avoid further losses. Moreover, management can decide to change the implementation choices made ex-ante, on the basis of information which only became available ex-post the investment decision. Management actions and decisions made during the life of an investment are valuable because they can push the pay-off of the initial investment closer to its upper possible threshold, rather than passively accept a probabilistic mean value.

The pay-off method utilizes fuzzy sets to determine the possibilistic, as opposed to the probabilistic, expected value of a given healthcare intervention. A fuzzy set is a class of elements with a continuum of grades of membership, characterized by a membership function which assigns to each element a grade of membership ranging between zero and one. In essence, the pay-off method replaces the probability distribution of a NPV outcome with a simpler triangular distribution, defined by a fuzzy set of three values:

a = corresponds to the base case mean NPV value

α= represents the distance between a and the minimum possible NPV value

β = is the distance between a and the maximum possible NPV value

The highest possibility (fully possible) is assigned to the base case and the lowest (near-zero) possibility to the minimum and maximum values of the distribution. The result is thus a triangular fuzzy distribution (A) that is equivalent to the fuzzy NPV of the project. The mean value of the positive values of the fuzzy NPV, E(A+), is the possibilistic mean value of the positive fuzzy NPV values, as shown graphically in Figure 1.

The possibilistic approach to uncertainty and the recognition of the economic value of the active role of management differentiate the pay-off pricing method for real options from the cost-effectiveness paradigm.

But would the pay-off method lead to different results compared to cost-effectiveness analysis?

To answer this question, we informed the pay-off model with cost-effectiveness data were derived from a full Bayesian study which evaluated the implementation and economic consequences of the HPV vaccination of 2, 3 and 4 cohorts of girls aged 12, 15, 18 and 25 years. Although the outcomes of the Bayesian model reported in the cost-effectiveness study could not be directly compared with any previous evaluation, the range of incremental cost per QALY gained for a single cohort of girls aged 12 years confirmed the cost-effectiveness dominance of immunising this cohort compared to the alternatives.

In contrast to the outcomes of a full Bayesian cost-effectiveness model, where the single cohort strategy is dominant, the pay-off method assigned the highest real option value to the two-cohort strategy, closely followed by the single cohort option. Figure 2 reports a summary of the pay-off valuation of the four immunization options.

The real option pricing of the four HPV vaccination strategies evaluated here offers new insights into the implementation of a national immunisation programme from the perspective of the National Health Service. The general acceptance of the use of cost-effectiveness analyses in the economic evaluation of healthcare can result in one of the most common errors in decision-making, commonly referred to as ‘frame blindness’, in which the wrong problem is addressed because a conceptual framework is created that results in a decision in which the best option is rejected.

The integration of cost-effectiveness analysis and real option valuation could improve the quality of policy decisions under conditions of uncertainty, due to the explicit recognition of the value of management in the implementation of healthcare programmes.



Prof. Giampiero Favato (Corresponding Author)


Giampiero Favato Figure-1Figure 1. Triangular distribution of fuzzy set A. The fuzzy set A is defined by three values: a (the best-case scenario NPV), α (the distance between the minimum and the best-case scenario NPV) and β (the distance between the maximum and the best-case scenario). The area between a-α represents the distribution of all possible negative NPV values while the opposite side, between 0 and a+β, shows the distribution of positive NPV values. The possibilistic mean value of the positive fuzzy NPV values E(A_+), is the fuzzy mean value of the NPV.


Giampiero Favato Figure-2Figure 2. Pay-off distribution of the four HPV vaccination strategies evaluated in the Real Options valuation. The left-hand side of the distribution shows the distance (α) between the minimum NPV value and the base-case scenario NPV (a) for the four vaccination options evaluated, as identified in the data legend located in the lower right-hand corner of the graph. The right-hand side of the distribution reports the distance (β) between the maximum NPV value and the base-case scenario NPV (a).
The dotted lines in the centre of the distribution show the real options value for each option, which is the possibilistic mean value of the positive fuzzy NPV values multiplied by the positive area of the fuzzy NPV over the total area of the fuzzy NPV. The highest Real Options value (€17,723, red dotted line) was attributed to the two-cohort vaccination, followed by the single cohort (€17,461, blue dotted line) and, more distantly, by the three- (green dotted line) and four- (orange dotted line) cohort vaccination (€16,440 and €13,576 respectively).

Exp Mol Pathol. 2013 Oct;95(2):166-73.

Balance of apoptotic and anti-apoptotic marker and perforin granule release in squamous intraepithelial lesions. HIV infection leads to a decrease in perforin degranulation.

Fernandes AT, da Rocha NP, Avvad E, Grinsztejn BJ, Russomano F, Tristão A, Quintana Mde S, Perez MA, Conceição-Silva F, Bonecini-Almeida Mda G.

Laboratory of Immunology and Immunogenetic in Infectious Diseases at Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.



Cell-mediated cytotoxicity plays an important role in the regulation to HPV-associated cervical intraepithelial neoplasia. HIV co-infection is related to poorer prognosis and more rapid clinical progression to cancer. We evaluated the presence of cervical inflammatory cells, apoptotic (Bax, Bcl-2, FasL, NOS2, perforin) markers and the degranulating expressing cell marker (CD107a) in low and high squamous intraepithelial lesions (LSIL and HSIL, respectively) from HIV-negative and -positive women. Higher percentage of cervical CD4+, CD8+ T cells and macrophage were observed in LSIL and HSIL groups when compared with control, especially in epithelium and basal layer of epithelium. However, progression from LSIL to HSIL did not change the frequency of inflammatory cells. HIV-infection lead to a reduction on cervical CD4+ T cell infiltration and an increased CD8+ T cell distribution in LSIL groups. A balance between pro- and anti-apoptotic protein expressions was verified. Bax-expressing cells were present in all groups and were rarely expressed in keratinocytes in the epithelium in LSIL and control groups, but notably decreased in HSIL group. However, its frequency was enhanced in the basal layer of the epithelium meanly in LSIL group. Bcl2-expressing cells in the epithelium and the stroma were enhanced in HSIL group when compared with LSIL group. HIV-infection did not interfere in both expressions NOS2 expression was located on keratinocytes in both LSIL and HSIL groups when compared with control group. There were few FasL cervical expressing cells in all groups. Indeed, perforin was identified in fewcervical cells. However, CD107a, a surfacemarker for cellular degranulationwas significantly higher in epithelium, basal layer of epithelium and stroma in LSIL and HSIL, respectively, when compared with control group. These results support that HIV infection may induce reduction on inflammatory cervical cell degranulation corroborating to carcinogenesis process. This is the first description on the role of HIV in downregulation of perforin degranulation in the cervical lesions and it might be related to carcinogenesis.

Published by Elsevier Inc.

KEYWORDS: Apoptosis, CTL, Cytotoxic dysfunction, HIV-infection, SIL, Uterine cervix

PMID: 23791892



Cervical cancer was declared the 4th cause of cancer death in 2010, and the second most common cause of cancer death in women. Infection with oncogenic types of HPV is the main causal factor for cervical cancer and its precursor lesion, squamous intraepithelial lesions (SIL). Many women are infected with HPV, but only a minority will develop SIL or cervical cancer. The majority of HPV infections induces low grade squamous intraepithelial lesion (LSIL) that in more than 90% of cases spontaneously regress and in about 10% eventually progress to high grade lesions (HSIL) and 1% frequently evolve to invasive cancer.

The mechanisms that control transition from latent infection to cervical cancer are unknown, although it is believed that progression to cervical cancer is characterized by: increased expression of viral oncoproteins E6 and E7 of high risk HPVs that deregulate the host cell growth cycle by binding and inactivating tumor suppressor proteins, cell cyclins, and cyclin-dependent kinases; integration of viral DNA into host genome, with disruption of E2 viral genes and host chromosomal loci; HIV co-infection; and cellular immunity that plays an important role in HPV infection, especially cytotoxic T cells.

Mechanisms of apoptosis are crucial for carcinogenesis control. Generally, apoptosis signaling is largely classified by the triggers involved into two pathways: the extrinsic, characterized by death receptor, as Fas and TNFR; and the intrinsic, characterized by mitochondrial dysfunction that is induced through the transcriptional or post translational regulation of apoptotic factors. The bcl-2 gene family acts as one of regulators of the apoptotic intrinsic pathway. The two most important apoptosis regulating proteins of this family are most likely a Bax and Bcl2, an apoptotic and antiapoptotic markers.

The central goal of this study was to identify cytotoxic as well as apoptotic markers in cervical uterine lesions from HPV and HPV/HIV co-infected women. Sixty women with SIL were consecutively enrolled from two cohorts from Fiocruz Clinical Care Units at Rio de Janeiro, Brazil. Cervical biopsies were snap-frozen in liquid nitrogen and mounted in OCT compound and serial cryostat sections were taken for immunohistochemistry. To determine the possible cytotoxic role of inflammatory cells, we analyzed the biomarkers FasL, perforin, CD107 (degranulation marker), Bax and Bcl2 in cervical cells by immunohistochemistry.

Balance of apoptotic and anti-apoptotic markers in HPV-infected keratinocytes determine the cervical lesion progression. So, we observed a decreased proapoptotic Bax-expressing cells in patients with more severe cervical lesion (HSIL) when compared with patients with benign lesions (LSIL) and women with no HPV lesions. Otherwise, anti-apoptotic Bcl2-expressing cells were observed meanly in HSIL women when compared with LSIL and control group (Figure 1). HIV infection do not interfered with these apoptotic markers expression. These results support an idea that HPV modulated to its favor the expression of proapoptotic and apoptotic markers in keratinocytes inducing pre-cancer lesions and further the carcinogenesis. These proteins can be used as prognostic biomarkers for pre-malign cervical lesion outcome.

Macrophages were the major inflammatory cervix cells during HPV and HIV infection. These cells produce nitric oxide from an induced nitric oxide synthase (NOS2) enzyme, an important microbicidal product. Interestingly, NOS2 expression was seen in keratinocytes located in the basal line of epithelium, instead macrophages in both LSIL and HSIL groups. This biomarker was highly expressed in both patients groups, meanly in LSIL, when compared with control group that rarely expressed NOS2. HIV-infection did not change NOS2 expression profile in the lesion (Figure 2a). The progression to more severe lesions reflect the loss of this important cellular regulatory factor. We are working to determine its role in carcinogenesis. However, NOS2 can not be used as a biomarker alone because it is induced by several inflammatory mediators during bacterial and viral infections.

Cytotoxic CD8 T cells are able to control viral infection through Fas and Perforin-based pathways, accounted for most of T-cell-mediated cytotoxicity. In cervical HPV-induced lesions, however, few cervical FasL-expressing cells in both LSIL and HSIL were observed (data not shown). Similarly, perforin expression was rare in both groups. Perforin is secreted after cytotoxic CD8 T cells encountered theirs viral targets. For monitoring the exocytosis of lytic granules in CD8+ T cells, the expression of CD107a was analyzed (Figure 2b). However, this marker was not present in the majority of inflammatory cervical cells. CD8+ T cells might have a dysfunction in the cervix, leading to fail to recognize HPV infected keratinocytes and further release of lytic granules. So, suggesting that HPV can somehow avoid the induction of an effective cellular immunity and escape of immune surveillance, leading to cervical lesion progression and further cancer development.

We believe that the cervical inflammatory microenvironment induced by HPV infection and host immune response can define the lesion progression. More studies are needed to determine prognostic biomarkers and to identify viral and host targets for new drugs discover.
figure-1Figure 1. Distribution of Bax- (A) and Bcl2-expressing cells (B) in uterine cervix from Control, LSIL and HSIL patients. The number of positive cells was demonstrated in epithelium, basal layer of epithelium , stroma and periglandular area.

Figure 2. Distribution of NOS2- (A) and CD107-expressing cells (B) in uterine cervix from Control, LSIL and HSIL patients. The number of positive cells was demonstrated in epithelium, basal layer of epithelium, stroma and periglandular area.

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