We assessed the incidence of genital warts in Germany and collected information on their management to estimate the annual cost of disease.Human papillomavirus (HPV) is a necessary cause of cervical cancer. HPV is also responsible for benign This was a multi-centre observational  study of genital warts in Germany. Data were collected from gynecologists, dermatologists, and urologists seeing patients with genital warts between February and April 2005. The number of patients with new and recurrent genital warts was used to estimate the incidence in Germany. We assessed resource use for patients with genital warts seen during a two-month period as well as retrospective resource use twelve months prior to the inclusion visit through a chart review. The mean costs of treatment of patients with genital warts from third-party payer and societal perspectives were estimated, and the total annual cost of genital warts was then calculated.For the incidence calculation 217 specialists provided information on 848 patients and 214 specialists provided resource use data for 617 patients to assess resource consumption. The incidence of new and recurrent cases of genital warts was 113.7 and 34.7 per 100 000, respectively, for women aged 14–65 years consulting gynecologists. The highest incidence was observed in women aged 14–25 years (171.0 per 100 000) for new cases and in women aged 26–45 years (53.1 per 100 000) for recurrent cases. The sample size for males was too small to allow a meaningful estimate of the incidence. The mean direct cost per patient with new genital warts was estimated at 378 euros (95% CI: 310.8–444.9); for recurrent genital warts at 603 euros (95% CI: 436.5–814.5), and for resistant genital warts at 1,142 euros (95% CI: 639.6–1752.3). The overall cost to third-party payers was estimated at 49.0 million euros, and the total societal cost at 54.1 million euros, corresponding to an average cost per patient of 550 euros and 607 euros, respectively.The societal burden and costs of managing and treating genital warts in Germany are considerable. A vaccination programme using the quadrivalent human papillomavirus vaccine could provide a substantial health benefit and reduce the costs associated with genital warts in Germany. It has been estimated that as many as 75% of the sexually active population (15–49 years of age) in the US have been infected with HPV during their lifetime .-19.16-19t-test. Statistical analyses were conducted using SAS version 8 .The cost per patient was calculated by multiplying the units of resources used by the unit costs. The mean cost per patient for each genital wart type was calculated and the 95% confidence intervals (95% CI) were obtained using non-parametric bootstrapping techniques because the distribution of costs was expected to be skewed. The impact of patient characteristics  and type of specialist on the overall costs were evaluated in a multivariable analysis. An F-test was performed to determine the overall significance of the model. The significance of each parameter in the model was determined using a The cost of illness for each type of genital warts in Germany was calculated by multiplying the estimated total number of patients per year by the mean annual cost per patient for each type. The costs for existing cases of unknown type were estimated using the weighted average costs for recurrent and resistant cases based on the observed proportions. The total annual cost of genital warts in Germany was then calculated as the sum of the costs for the four types. The analysis was performed from both the third-party payer  and societal  perspectives. The cost for specialist visits, diagnostic tests, medication, and procedures were included in the direct medical costs, and the cost of lost work days was considered in the indirect cost. The cost for retrospective and current resource use was included in the costs for patients with resistant and recurrent lesions. For patients with new genital warts, the costs were based on the current visit and the prescriptions by the physician during the consultation. The overall cost to third-party payers and the total societal cost included both women and men, and all specialists.Between January and March 2005, 401 specialists were invited by telephone to participate in the epidemiological survey on genital warts. Of these, 217 agreed to participate: 135 gynecologists, 55 dermatologists, and 27 urologists. Data for 848 patients with genital warts were collected for the study, including 508 new patients (incident) and 340 patients with existing genital warts  Table . The majA total of 214 physicians participated in the chart review study, including 129 gynecologists, 59 dermatologists, and 26 urologists. More than 90% of the specialists were office-based, and the others were based in hospitals. Most of the specialists (85%) worked in their own medical practice. Data for 621 patients were collected; data for four of these patients were excluded because they did not meet the age criteria (n = 3) or had a treatment duration of over 12 months (n = 1).The study sample (n = 617 patients) included 384 women (62%) and 233 men (38%). The mean age was 32.0 ± 10.0 years. There was no significant difference in age between the patients with new (32.2 ± 10.1 years) and existing (31.6 ± 9.9 years) genital warts. Treatment follow-up time was not available for 12 of the 189 patients with existing genital warts. The remaining 177 patients had a mean follow-up of 6.7 ± 4.3 months. Approximately half (48%) of the patients in the study had an elementary or primary school education, 28% had a secondary or high school education, and 12% had a college or postgraduate degree. Of the 352 patients with data available, the majority (95%) reported having had three or fewer new sexual partners in the previous year: 13.9% had no new partner, 52.0% had one new partner, 20.7% had two new partners and 7.9% had three new partners and 5.5% had more than three new partners. Most of the patients (76%) reported that they had not had a sexually transmitted infection previously.Sixty-nine percent n = 428) of the patients were newly diagnosed with genital warts; 15% had recurrent genital warts (n = 92), 6% had resistant genital warts (n = 36) and for 10% (n = 61) the type of existing genital warts was not specified  saw a gynecologist, 117 (27%) a dermatologist, and 57 (13%) an urologist  the most frequent diagnostic tests were visual examination (36.7%), Pap smear tests 15.7%), colposcopy (12.6%) and biopsy (10.7%). For those with recurrent (n = 92) and resistant genital warts (n = 36) visual examination was by far the most frequent diagnostic test (94.6% and 97.2% respectively), followed by Pap smear tests (32.6%), and biopsy and colposcopy (26.1% for each) and 19.6% had histological tests. Imiquimod, podophyllotoxin solution, and podophyllotoxin cream were the most frequently self-administered medications for all patients (data not shown). There was no difference in the frequency of use of these therapies for new, recurrent, and resistant genital warts. Table .7%, colpAll costs were highest for resistant genital warts: 1,141.8 euros (95% CI: 639.6–1752.3) for the mean annual direct cost and 1,369.8 euros (95% CI: 830.1–1979.1) including indirect costs. Direct mean cost for recurrent genital warts were 602.7 euros (95% CI: 436.5–814.5) and 732.91 euros (95% CI: 514.6–961.9), including indirect costs and 377.6 euros (95% CI: (310.75–444.88) for new genital warts (included direct cost only). Table The total estimated annual direct cost for new, recurrent, resistant, and existing genital warts of unknown status in Germany was 20.8 million euros, 10.0 million euros, 5.7 million euros, and 12.0 million euros, respectively. Including indirect costs the total expenditures rose to 12.7 million euros, 6.7 million euros, and 14.1 million euros for recurrent, resistant, and existing genital warts of unknown status, respectively. On the basis of these calculations, the total annual cost of genital warts in Germany was estimated at 49.0 million euros from a third-party payer perspective (indirect costs excluded) and 54.1 million euros from a societal perspective (indirect costs included), corresponding to an average cost per patient of 550 euros and 607 euros, respectively.P < 0.05) , type of wart, and location of wart  as factors significantly affecting the total cost of genital warts (5) Table . Gender,The incidence of genital warts was 171/100 000 in 14–25 year old women and 157/100 000 in 26–45 year old women. This study confirms that genital warts are very prevalent in young to mid-adult women in Germany. Furthermore, the results indicate an annual health and economic burden to the German health care system of at least 89 000 cases of genital warts and 54 million euros in associated costs.The overall incidence of new genital warts estimated for Germany is lower than those reported in women in the United Kingdom and France ,22. ResuA report on the management of genital warts in the UK estimated that the mean direct cost per new and recurrent patient was £180 (266 euros) and £229 (338 euros), respectively . In The The study has several limitations. Although the physicians who participated in this study were representative in terms of geographic distribution and type of practice  we cannot exclude that physicians who did not participate were more or less likely to see patients with genital warts. Also some of the physicians participating in the study reported no patients consulting for genital warts during the 2-month study period and some could not provide an accurate estimate of the number of patients with genital warts in their practice. In addition, we could not verify if the patients with genital warts seen by the physicians participating in the study were representative out of all patients with genital warts in Germany. In addition, we assumed that all patients with genital warts consult a physician; however, it is possible that some patients with genital warts decided not to seek medical care because they do not consider that the condition is serious enough to seek medical care. If so, ascertainment bias may have resulted in an underestimation of the true incidence of genital warts in our study. Our results show the incidence of patients seeking medical care for genital warts. These biases may have resulted in either an over- or underestimation of the true incidence of genital warts in Germany.A second limitation is the extrapolation of the total number of men with genital warts. The incidence of genital warts in men visiting dermatologists or urologists was not calculated because too few data on males with genital warts were available to provide a reliable estimate of the incidence. From data obtained from the genital warts surveillance system in the UK it is known that the incidence for genital warts is comparable for men (146 per 100 000 population) and women (122 per 100 000 population) [Lastly, our estimate of the total cost of genital warts in Germany is likely to be underestimated. Annual resource consumption data was collected only for existing patients. For new patients, only the costs related to the first visit were included, and no indirect costs were included. Moreover, the estimated number of men with genital warts is likely to be underestimated, resulting in an underestimation of the associated cost.Recent results from clinical trials of prophylactic vaccination of women between 16 and 24 years of age with a quadrivalent HPV  L1 virus-like particle vaccine (Gardasil) have shown 100% vaccine efficacy for the co-primary composite endpoints; incidence of genital warts, vulvar or vaginal intraepithelial neoplasia or cancer and incidence of cervical intraepithelial neoplasia, adenocarcinoma in situ or cancer associated with HPV type 6, 11, 16 or 18 (per-protocol susceptible population) . This va® in Europe. PH declared that he has received honorarium for lectures and clinical studies from Sanofi Pasteur MSD, GSK; Digene and Cytyc. KJL declared that she worked for MAPI Values who were contracted by Sanofi Pasteur MSD to analyse the study data. FG and KUP declared that they have received honorarium from Sanofi Pasteur MSD and GSK for speaking at several scientific meetings and for acting as scientific consultants.JGB, SB and EL previously worked for Sanofi Pasteur MSD, who commercializes GardasilPH, JGB, FG, SB, and KUP were involved in the study conception, JGB and EL were involved in the coordination, KJL performed the data analyses, all authors were involved in the interpretation and validation of the results, PH, JGB and KUP were involved in the drafting of the manuscript and all authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Mulligan's mobilisation techniques are thought to increase the range of movement (ROM) in patients with low back pain. The primary aim of this study was to investigate the application of the Mulligan's Sustained Natural Apophyseal Glide (SNAG) technique on lumbar flexion ROM. The secondary aim was to measure the intra- and inter-day reliability of lumbar ROM employing the same procedure.3and L4 spinal levels with active flexion in sitting by an experienced manual therapist. Three sets of 10 repetitions at each of the two spinal levels were performed. The sham mobilisation was similar to the SNAG but did not apply the appropriate direction or force. Lumbar ROM was measured by a three dimensional electronic goniometer (Zebris CMS20), before and after each technique. For the reliability, five measurements in two different days (one week apart) were performed in 20 healthy subjects.49 asymptomatic volunteers participated in this double-blinded study. Subjects were randomly assigned to receive either SNAG mobilisation (n = 25), or a sham mobilisation (n = 24). The SNAG technique was applied at the Lt tests yielded no statistically significant results in ROM between groups (p = 0.673). Furthermore no significant within group differences were observed: SNAG (p = 0.842), sham (p = 0.169). Intra- and inter-day reliability of flexion measurements was high  indicating acceptable clinical applicability.When both interventions were compared, independent While the Zebris proved to be a reliable device for measuring lumbar flexion ROM, SNAG mobilisation did not demonstrate significant differences in flexion ROM when compared to sham mobilisation.Current Controlled Trials NCT00678093. It is common to find stiffness and reduced lumbar range of movement (ROM) in clinical presentations of mechanical low back pain (LBP) with a limited ability to perform flexion of the trunk -5. MulliAmong the SNAG's basic principles of clinical management is an immediate reduction or cessation of pain and an increase in range of motion (ROM) -15. The Measurement of ROM as an outcome measure for LBP patients is a common procedure both in clinical practice and in research providing clinicians with insight into the patient's overall physical profile -22. ROM Given the above, the primary aim of this study was to explore whether an immediate increase in flexion ROM occurs following the application of a lumbar SNAG technique in an asymptomatic sample. A secondary aim was to investigate the reliability of the Zebris equipment utilized for measuring the lumbar ROM in the study.The intervention component of this study was a prospective randomized double-blinded controlled study investigating the effect of SNAG on lumbar flexion ROM of a healthy sample of volunteers. A reliability study of measuring lumbar flexion ROM with the three dimensional (3-D) electronic goniometer Zebris CMS20, preceded the main study. Ethical approval was obtained from the Ethical Committee of the Technological Educational Institute (T.E.I.) of Lamia, Greece.d = 3.8 . Thus, Forty-nine asymptomatic volunteers  were recruited into the interventional component of the study. Their ages ranged between 18 and 32 years old (mean: 21 years). Exclusion criteria included parameters that could influence ROM i.e. no current or previous (within the last 2 years) LBP episodes, no spinal surgery or other spinal pathology  -45. MoreLumbar ROM was measured with the Zebris CMS20 ultrasound-based motion analysis system . This 3-D lumbar motion equipment consists of two specially designed belts  each attached to a series of three miniature ultrasound transmitters that are fixed to the subject's torso Figure . For isoROM measurements were performed by a physiotherapist experienced in the use of Zebris equipment. The therapist was blinded to the type of intervention. All measurements took place at least two hours after waking up in the morning in order to overcome the initial diurnal stiffness of the spine and circadian variation ,52,53. FAn accredited manual therapist with 7 years of clinical experience in treating LBP patients and substantial training in the Mulligan concept performed the manual techniques. Following the first set of ROM measurements, each participant was randomly assigned into one of the two intervention groups (SNAG or sham) by selecting one of two identical cards representing each technique. Neither the participant, nor the physiotherapist performing the measurements was aware of the selected technique. The techniques were applied by the manual therapist on L3 and L4 lumbar levels. These particular levels were chosen because it has been found in cadaver segmental motion studies that L4/5 has stiffer elements compared to other motion segments -59 and mThe sham technique appeared very similar to the SNAG but the application of the optimum direction and the appropriate amount of force were absent. In particular, the therapist's hand was just placed (without exerting any pressure) over the relevant spinous process and the direction of this very 'gentle' pressure of the hand was downward . These two factors (direction and force) are considered to play a vital role in the success of the technique ,13. NoneFor the reliability measurements, all procedures undertaken  were identical with the interventional component of the study. Each subject was examined on five occasions; with three tests were performed the same day (intra-day reliability), and the other two tests performed one-week later (inter-day reliability). On each occasion, following a practice trial, three active lumbar flexion movements were performed by each subject at their normal, preferred speed ,54,60-621,1), standard error of measurement (SEM) and the smallest detectable difference (SDD) reliability indices were used for all parameters being tested. The SDD represents a clinically applicable percentage figure for the amount of change needed to detect a true change in a subject's performance [As all data were normally distributed an independent sample test was utilized for detecting statistical differences in ROM between the groups (SNAG versus sham). The paired tests were utilized to explore statistical differences in lumbar ROM within each group. Levels of significance were set at 5%. For the reliability study, the intraclass correlation coefficient  and 24 to the sham group . No significant differences were noted between the groups' demographic and baseline ROM data , nor the sham group (p = 0.169). Statistical results and confidence intervals (CI) following the interventions are illustrated in Table For the ROM measurements, only the second and third recordings from each repetition were used in order to avoid any learning effect often observed between the first and second repetition . A mean For the reliability study, the anthropometric characteristics of the subjects are presented in Table The study was designed to investigate the immediate mechanical effect of a Mulligan's SNAG technique on lumbar flexion ROM since it is suggested that flexion is the movement most often affected in LBP ,2,24. Thfirst (rather than randomly applying placebo and intervention treatments), the study's outcome could have been affected (by this 'order').These results are in accordance with previous literature demonstrating non-statistically significant results in ROM when applying manipulative therapy techniques in asymptomatic subjects ,68. PettSo far, research regarding MWMs' effectiveness in increasing ROM is limited to case studies where only peripheral joints are evaluated ,71. OnlyThe protocol of therapeutic interventions may be another factor to consider in evaluating the results of the current study. Although there is no suggested regime on the number of repetitions that would benefit LBP patients, Mulligan  suggestsThe use of an asymptomatic population remains a potential limitation to this study. A sample of LBP patients may have been more appropriate in reflecting clinical practice. With symptomatic subjects, it is hypothesized that MWMs reduce pain and subsequently restore ROM through various inter-acting mechanisms including articular, peri-articular, neurophysiological, endocrine, and psychological effects ,77. ReduIn terms of clinical significance of the study, it is emphasized that ROM measurement may be a useful indicator of monitoring patient progress  -22,80,81Regarding the reliability part of the study, the results demonstrated excellent intra- (ICCs = 0.89–0.90) and inter-day (ICCs = 0.82) reliability in lumbar measurements with the Zebris. Intra-day flexion measurements are comparable with previous reports, and in most cases our reliability is considerably higher with lower measurement errors ,33,83-85In this prospective randomized double-blinded study, SNAG mobilizations applied at two lumbar levels in asymptomatic subjects did not demonstrate significant differences in the lumbar flexion ROM, when compared to a placebo group. Focus for further research should involve symptomatic subjects with reported (and/or measurable) restriction in lumbar ROM, and explore the global effect of MWMs in terms of biomechanics and pain. In terms of the reliability of the measuring equipment, although the present procedure yielded excellent intra- and inter-day reliability measurements, indicating good clinical applicability, improvement of the Zebris fixation system is recommended.The authors declare that they have no competing interests.MM carried out the ROM measurements in the interventional component of the study, was involved in the acquisition and interpretation of data, and wrote the first draft of the manuscript. EB served as project lead, was responsible for the conception and design, carried out the manual interventions, contributed in the analysis and interpretation of the interventional component and has been involved in drafting the manuscript. NS contributed substantially to the conception of the reliability component of the study, its data acquisition and analysis and was involved in drafting the manuscript. KP carried out the acquisition, analysis and interpretation of data in the reliability component of the study. JO helped in revision of the manuscript.The pre-publication history for this paper can be accessed here:


Leucine-rich repeats (LRRs) are present in more than 6000 proteins. They are found in organisms ranging from viruses to eukaryotes and play an important role in protein-ligand interactions. To date, more than one hundred crystal structures of LRR containing proteins have been determined. This knowledge has increased our ability to use the crystal structures as templates to model LRR proteins with unknown structures. Since the individual three-dimensional LRR structures are not directly available from the established databases and since there are only a few detailed annotations for them, a conformational LRR database useful for homology modeling of LRR proteins is desirable.We developed LRRML, a conformational database and an extensible markup language (XML) description of LRRs. The release 0.2 contains 1261 individual LRR structures, which were identified from 112 PDB structures and annotated manually. An XML structure was defined to exchange and store the LRRs. LRRML provides a source for homology modeling and structural analysis of LRR proteins. In order to demonstrate the capabilities of the database we modeled the mouse Toll-like receptor 3 (TLR3) by multiple templates homology modeling and compared the result with the crystal structure..LRRML is an information source for investigators involved in both theoretical and applied research on LRR proteins. It is available at  Leucine-rich repeats (LRRs) are arrays of 20 to 30 amino acid long protein segments that are unusually rich in the hydrophobic amino acid leucine. They are present in more than 6000 proteins in different organisms ranging from viruses to eukaryotes . The strThe LRR sequences can be divided into a highly conserved segment (HCS) and a variable segment (VS). The highly conserved segment consists of an 11 or 12 residue stretch with the consensus sequence LxxLxLxxN(Cx)xL. Here, the letter L stands for Leu, Ile, Val or Phe forming the hydrophobic core, N stands for Asn, Thr, Ser or Cys, and x is any amino acid. The variable segment is quite diverse in length and consensus sequence, accordingly eight classes of LRRs have been proposed ,5: 'RI-lThe discrepancy between the numbers of structure-known LRR proteins and the structure-unknown ones triggered studies focusing on the homology modeling of LRR proteins -8. HomolStructure-known LRR proteins were extracted from the Protein Data Bank (PDB)  release 1. A LRR begins at the beginning of the highly conserved segment (HCS) and ends at the end of the variable segment (VS) (just before the HCS of the next LRR).2. The HCS of a LRR must pose a typical conformation, i.e. a short β-sheet begins at about position 3 and a hydrophobic core is formed by the four L residues at position 1, 4, 6, and 11.The LRRs were then manually classified according to the consensus sequences ,5. In adDuring the LRR identification and classification all sequence insertions longer than 3 residues were annotated. About one tenth of entries have insertions longer than 3 residues while few entries have deletions, which suggests that the evolution of LRRs may prefer insertion to deletion.The LRRML release 0.2 contains 1261 LRR entries from 112 PDB structures. Among them 548 LRRs are distinct on sequence level, indicating that different molecules can share identical LRRs. By superimposition, we found that they also have highly similar structures. This fact enhances the confidence in modeling LRR proteins using multiple LRR templates. A histogram of entry length distribution  was standardized in the 90s and is well established as a format for hierarchical data. It can be queried and parsed more easily by application programs. Therefore, more and more biological databases use the XML as data saving format and database management system (DBMS) -19. LRRM1. The sequence information (XML tag <l:Sequence>): amino acid sequence and sequence length.2. The classification information (XML tag <l:Type>): class name and consensus sequences.3. The sequence partitions (XML tag <l:Regions>): amino acid sequence, position, length and insertion of HCS and VS.4. The corresponding PDB sources (XML tag <l:Sources>): ID, chain, LRR number and classification of the source PDB entries; serial number, position, DSSP  secondar2O6S is shown in Figure An example describing the LRR3 from PDB entry The entire database can be browsed by LRR IDs or by PDB IDs. When browsing, the entries appear in a summary table containing at first ID, type and sequence. Clicking on an ID opens an XML Stylesheet (XSLT)  converte...' is treated by the server as path to a database collection. Also, request parameters can help select any required elements. For example, '_query' executes a specified XPath/XQuery; the URL "" returns all the S type LRRs.The DBMS provides a REST-style application programming interface (API) through HTTP, which supports GET and POST requests. A unique resource identifier (URI) 'LRRML was designed as a tool for template selection in homology modeling of LRR proteins. Traditionally, the template used in homology modeling is one or more full length protein structures obtained via similarity search. Nevertheless, due to the different repeat numbers and arrangements of LRRs, the sequence identity between the target and the full length template is usually not high enough for homology modeling. With LRRML the most similar structure-known LRR can be found for each LRR in the target sequence as a local template. The combination of all local templates through multiple alignments helps to achieve a high sequence identity to the target.3CIG) was 98.6%  ectodomain. We assumed that the structure of mouse TLR3 ectodomain were unknown and excluded the LRRs of mouse/human TLR3 ectodomain from LRRML. Through similarity search the optimal template for each of the 25 LRRs in mouse TLR3 was found. The sequence identity between each LRR pair (target/template LRR) is listed in Table % Figure . The mou% Figure , indicatA specialised conformational leucine-rich repeats database called LRRML has been developed. It is supported by an XML database management system and can be searched and browsed with either an easy-to-use web interface or REST like interface. The interface is suitable for most graphical web browsers and has been tested on the Windows, Mac and Linux operating systems. LRRML contains individual three-dimensional LRR structures with manual structural annotations. It presents useful sources for homology modeling and structural analysis of LRR proteins. Since the amount of structure-determined LRR proteins constantly increases, we plan to update LRRML every 2 to 3 months..This database is freely available at TW and JG drafted the manuscript, extracted the data, compiled the database, wrote the code for the web interface and performed the statistical analysis. FJ, WMH, RWS and SCR conceived of the study, built the database server, participated in the database design and coordination and helped to draft the manuscript. TW and JG should be regarded as joint first authors. All authors read and approved the final manuscript.The document type definition (DTD) file of LRRML.Click here for fileThe XML Stylesheet (XSLT) of LRRML.Click here for file


Increase in reactive oxygen metabolites (ROM) and free radicals is an important cause of cell injury. In this study, we investigated whether determination of ROM in pleural fluids of patients with malignant and non-malignant pleural effusions can be used as a tumor marker indicating malignant effusions in the differential diagnosis.t-test and the Mann-Whitney U test were used in order to detect differences between groups for descriptive statistics in terms of pointed features. The statistical significance level was set at 5% in computations, and the computations were made using the SPSS (ver.13) statistical package programSixty subjects with exudative pleural effusion and 25 healthy individuals as the control group were included in the study. Of the subjects with pleural effusion, 50% were malignant and 50% were non-malignant. ROM was studied in the pleural fluids and sera of the subjects with pleural effusion and in the sera of those in the control group. The ROM values of smokers and non-smokers were compared in each group. The Student’s P = 0.0001), and the sera ROM values of patients with non-malignant pleural effusion were significant compared to the control group (P = 0.0001), and the ROM values of smokers were significant compared to non-smokers in each of the three groups (P = 0.0001).It was determined that the difference between the ROM values of subjects with malignant and non-malign pleural effusions and the sera of the control group was significant in the malignant group compared to both groups (These findings indicate that sera ROM levels are increased considerably in patients with exudative effusions compared to that of the control group. This condition can be instructive in terms of serum ROM value being suggestive of exudative effusion in patients with effusions. Furthermore, the detection of pleural ROM values being significantly higher in subjects with malignant pleural effusions compared to non-malignant subjects suggests that ROM can be used as a tumor marker in the differential diagnosis of pleural effusions of unknown origin. Pleural effusions are mirrors of systemic diseases. Diseases of every system or organ can affect the pleura. For this reason, in the evaluation of pleural effusions, not only pulmonary or pleural diseases, but also pathologies below the diaphragm, systemic diseases and lymphatic system diseases must be taken into consideration.In a patient in whom pleural effusion is detected on the chest X-ray, the differentiation between transuda-exuda must primarily be made. The rate of true positivity for exudative fluids is 99.5% and the rate for transudative fluids is 75% in the assessment according to the Light criteria defined by Dr. Richard Light in 1972. comprised 90% of the malignant cases. Two (6.67%) cases were lymphomas (non-hodgkin) and one (3.33%) case was ovarian tumor . Diagnosis of malignant effusion cases was made by pleural fluid cytology (70%), histopathological analysis of pleural biopsies (23.33%) and pleural biopsy + cytological analysis (6.67%). The amount of pleural effusion was between 500 and 2000 ml in 75% of the patients. While the amount of fluid was below 500 ml in 15% of the cases, there was massive pleural effusion in the remaining 10%. While there was a lymphocyte predominance of 90% in the pleural fluid, eosinophilia (>%10) was detected in 15% of the patients. The mean protein concentration was 4 g/dl, with a range of 1.5 and 8.0 g/dl. The fluids of the patients with malignant effusions were detected as serous in 30% of cases, serousanginous in 40% of cases and hemorrhagic in 30% of cases. The mean erythrocyte count was detected as around 40,000/mmIn 12 (57.15%) of the patients in whom pneumonia was detected by chest X-ray and thoracic tomography, the width of fluid was found to be larger than 10 mm, glucose level was >40 mg/dl, pH was >7.2 in pleural fluid analysis and gram staining and culture were negative. In nine (42.85%) cases, the width of the fluid was found to be larger than 10 mm, pH <7.0, glucose < 40 mg/dl, gram staining and culture were positive, and marked pus was not present.Bacillus tuberculosis was detected in pleural fluids and pleural biopsy samples of cases with tuberculous pleurisy. In the pleural fluid analysis, the pH was between 7.30 and 7.40 and below 7.00 in approximately 20% of the patients. Pleural fluid glucose concentration was above 60 mg/dl in 71.43% of cases and below 30 mg/dl in 28.57% of cases. In the pleural fluid analysis of the patients, there was a slight lymphocyte predominance in more than 50% of the cases.Streptococcus pneumoniae) were detected on gram staining of pleural fluid in one patient with empyema, bacteria (Haemophilus influenzae) were isolated from the pleural fluid culture of the other patient. The same microorganism grew in blood cultures of the patient in whom bacteria were detected in the pleural fluid. The cell number was found to be 42,890 ± 56,816/mm3, protein was 4466 ± 1524 g/dl (1.5–8.7 g/dl), glucose was 43.1 ± 39.5 mg/dl (0–96 mg/dl), lactate dehydrogenase was 7851 ± 10503 U/l (378 – 28180 U/l), and the rate for polymorphonuclear leukocytes was found as 90.5 ± 14.5% in the pleural fluid analysis of these patients.Although bacteria  including reactive oxygen radicals  is a commercial kit that provides total measurement of oxidative stress and it was studied with Technicon RA-XT brand autoanalyzer.t-test and the Mann-Whitney U test were used for determination of the differences between groups in terms of these features. Statistical significance was accepted as 5% in calculations, and the calculations were made using the SPSS (ver. 13) statistical package program.Descriptive statistics for the mentioned characteristics were defined as mean, standard deviation, minimum and maximum values. The Student P = 0.0001) [It was found that ROM values were significantly higher in the malignant group compared to the non-malignant group and the control group, and it was higher in the non-malignant group compared to the control group , and dif 0.0001) .P = 0.0001) [When ROM values in pleural effusions were compared, it was found that the mean ROM values of malignant effusions were higher than that of the non-malignant group , and the 0.0001) .P = 0.0001, P = 0.004) [In the statistical analysis of pleural and serum ROM values according to 22 (73.34%) smokers and 8 (26.66%) non-smokers in the malignant group, the difference between the two groups was found to be statistically significant (= 0.004) .P = 0.0001) [A significant difference was found between the two groups in the statistical analysis of pleural and serum ROM values of 13 smokers (43.34%) compared to 17 non-smokers (56.66%) in the non-malignant group ( 0.0001) .P = 0.0001) [A statistical significance was found between the two groups in the statistical analysis of 9 (36%) smokers compared to 16 (64%) non-smokers in the control group ( 0.0001) .Pleural effusion can arise from pulmonary or systemic diseases. Once a pleural fluid sample is obtained, differentiation of transudate-exudate must primarily be made.Unlike exudative pleural effusions, an increase in reactive oxygen species in the pleural fluid is not anticipated as transudative pleural effusions are not related to local pleural pathologies, and develop related to the association between hydrostatic and oncotic pressures, whereas oxidative stress products increase in diseases causing both malignant and non-malignant exudative effusions as the pleural cavity is involved and mesothelial cells are held responsible for releasing oxidants. ROM in tFree oxygen radicals are defined as molecular structures including one or more unmapped electrons in their external orbits. Oxidant destruction caused by free oxygen radicals developing as a result of reduction of a single electron plays a role in the pathogenesis of diseases by being present in many events such as ischemia, hyperoxygenation and tissue inflammation.18There are many studies investigating the different parameters in the differential diagnosis of malignant and non-malignant pleural effusions. Studies in which tumor markers and tumor growth factors are used are popular. Although the contribution of these parameters to the diagnosis is uncertain, it is reported that combination of cancer antigen  and carcinoembryonic antigen (CEA) indicates a probable malignant mesothelioma. CEA is found to be high in malignant pleural effusions related to squamous and adenocarcinoma; CA-3 is found to be high in pleural fluids caused by breast cancer.In studies conducted with vascular endothelial growth factor (VEGF), it has been reported that this mediator is substantially produced in the diseased pleural tissue and plays a role in pleural fluid development, angiogenesis and vascular permeability, and is found in high amounts in non-small cell lung cancer, mesothelioma and breast cancer.Insulin-like growth factor values have also been reported to be high in malignant and non-malignant pleural effusions.21Recent studies investigating the outcomes of high ROM have established that free radicals are present in the pathophysiology of many diseases. Free radicals are known to be involved in cancer, inflammation injury, aging and chemical toxicity. Along with their effects in carcinogenesis, important roles of oxygen metabolites in inflammation defined as the reaction of live vascularized tissue against local injury have been discussed and depending on this, the increase in their levels is anticipated in diseases resulting in non-malignant effusions.Our current knowledge is that pleural ROM values increase in non-malignant exudative effusions along with malignant diseases. There are studies in the literature indicating that oxidative stress increases in non-malignant patients. Oxidative stress is reported to increase in active pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumonia, in patients exposed to asbestos and in smokers.24P = 0.0001) [In our study, directly ROM in sera and pleural fluid were assessed and ROM values in patients with malignancies, the majority of which comprised lung cancer and patients with non-malignant diseases, were found to be higher than in the control group, consistent with the literature–28 (P =  0.0001) .Despite the presence of many studies indicating that the serum ROM level increases in patients with malignancies,–28 studi1629P = 0.0001) [We did not encounter a study on the use of pleural fluid ROM values in the differential diagnosis in malignant and non-malignant cases. In our study, we found the pleural fluid ROM values in malignant cases to be significantly higher than that of non-malignant cases ( 0.0001) .P = 0.0001) [Tables There are studies indicating that ROM is affected by smoking, and that ROM levels in smokers are higher compared to non-smokers. In these studies, the increase in ROM in smokers is explained by cigarettes being the source of the oxidants.32 In our) Tables ‐6.In conclusion, our study shows that serum ROM values are considerably increased in patients with exudative effusion compared to the control group. This condition suggests that serum ROM values can be instructive in terms of exudative effusion in patients with effusion. Furthermore, detection of significantly high pleural ROM values in malignant pleural effusion compared to non-malignant cases makes ROM appropriate for use as a tumor marker in the differential diagnosis of pleural fluids of unknown origin.


Diagnostic and epidemiologic virology laboratories have in large part traded conventional techniques of virus detection and identification for more rapid, novel, and sensitive molecular methods. By doing so, useful phenotypic characteristics are not being determined. We feel that the impact of this shift in emphasis has impaired studies of the biology of viruses. This position paper is a plea to the scientific and administrative communities to reconsider the importance of such information. We also suggest a revised paradigm for virus isolation and characterization and provide a rationale for accumulating biologic (phenotypic) information.


When a virus infects a cell, it must contend with a hostile environment and host machinery that is intrinsically antiviral. One of the hallmarks of herpes simplex virus (HSV) infection is the dramatic reorganization of the infected cell nucleus leading to the formation of large globular replication compartments in which gene expression, DNA replication, and encapsidation occur . During The formation of replication compartments follows an ordered assembly process, resulting in a drastic remodeling of the nucleus Remodeling of the nucleus also involves the reorganization of the cellular PQC machinery, including cellular chaperone proteins  and components of the 20S proteosome Herpesviruses have also evolved a complex relationship with host DNA damage response pathways  and the single-strand binding protein (ICP8) interact with each other, are recruited to replication compartments, and are essential for efficient virus production (During the earliest stages of viral infection, HSV transforms the cellular environment from one that is hostile to virus infection to one that supports virus growth. Work in this area has only scratched the surface in terms of defining the mechanisms by which ICP0 and other viral proteins manipulate cellular pathways and create environments that promote lytic infection. Elucidating how cellular homeostatic pathways limit viral infections will be important to our understanding of the delicate balance between lytic and latent infection and to aid in the development of new antiviral therapies.


West Nile virus (WNV) was isolated from a patient who developed encephalitis while undergoing treatment with CHOP  and rituximab for a non-Hodgkin B-cell lymphoma. Both standard reverse transcription–polymerase chain reaction (RT-PCR) and Taqman RT-PCR established the diagnosis of WNV infection from cerebrospinal fluid (CSF). Several whole blood samples and one serum sample underwent further testing. CSF and serum samples were negative for WNV antibody; however, all samples were positive by both RT-PCR assays. Infectious virus was recovered from a blood sample, and its identity was confirmed by using a WNV-specific immunofluorescence assay. The complete WNV genomes determined from CSF and from the virus isolate adapted from cell culture were the same. The results represent the first complete WNV genome sequence obtained directly from human CSF and the first time that infectious WNV has been recovered from a patient with encephalitis in North America. West Nile virus (WNV), an arthropod-borne virus, is a member of the Japanese encephalitis virus serocomplex of the genus Flavivirus, family Flaviviridae  chemotherapy plus rituximab , to be followed by involved field radiation. After the first cycle of chemotherapy in July 2001, neutropenia developed in the patient after both the second and third cycles of chemochemotherapy. The third cycle of chemotherapy was administered on September 11, 2001. Four days later, she was treated with oral levofloxacin for low-grade fever. On day 7 after chemotherapy, she was admitted to the hospital with fever, cough, chills, rhinorrhea, joint aches, decreased appetite, lethargy, and lightheadedness. She had no recollection of any ill contacts or insect bites. She was a resident of southern Nassau County, New York, and did not have a history of travel. Physical examination was within normal limits. The patient was neutropenic; G-CSF was continued, and she was given cefipime and gentamicin. One day after admission, she continued to have fever and experienced mild headaches that responded to acetaminophen. Urine cultures showed penicillin-sensitive enterococci, and blood cultures were negative. Two days after admission, the patient continued to have fever, headaches, and dizziness. A computed tomography (CT) scan of the brain revealed no acute cerebral processes. On the 3rd day after admission, the patient was noted to be confused. Further deterioration of mental status was noted, with incomprehensible speech, but she was able to follow commands. Arterial blood-gas analysis showed an acute respiratory acidosis pattern, and the patient was subsequently intubated and transferred to the intensive care unit. The patient had a hypotensive episode secondary to atrial flutter, which required cardioversion for stabilization. Ceftriaxone and ampicillin were added to the antibiotic coverage, and antifungal treatment was also initiated with lipid complex amphotericin B. After the patient’s hematologic parameters improved, G-CSF was discontinued on day 9 of hospitalization, and the patient continued to be afebrile. Because of the clinical picture of encephalitis and because the patient lived in an area where WNV was endemic, lumbar puncture was performed on day 8, and a CSF specimen was sent to the New York State Department of Health laboratories for comprehensive PCR testing. Renal tubular necrosis developed in the patient, leading to acute renal failure by day 10, with further deterioration of her mental status. Dialysis was initiated on day 16, and antibiotic therapy was discontinued on day 17 as the patient remained afebrile and the neutropenia was resolved. The patient remained unresponsive, staphylococcal septicemia developed, and she died on day 35 of hospitalization. Autopsy showed a small focus of perivascular lymphocyte cuffing in the mamillary bodies of the brain, consistent with viral encephalitis. No evidence of residual lymphoma was found.Human herpesvirus 4), enteroviruses, and the following arboviruses: Eastern equine encephalitis, California serogroup , Powassan virus (POWV), St. Louis encephalitis virus (SLEV), WNV, and Cache Valley virus.The PCR Laboratory associated with the Virology Diagnostic Services, Wadsworth Center, New York State Department of Health, has developed a panel of PCR and RT-PCR assays that allow tests on CSF and brain tissue for a wide range of viruses associated with human central nervous system (CNS) infections. Specifically, the test battery includes herpes simplex viruses (types 1 and 2), varicella-zoster virus, cytomegalovirus, Epstein-Barr virus  according to the manufacturer’s instructions. Briefly, RNA was first transcribed into cDNA with random primers , and an aliquot of this cDNA (5 μL) was used in PCR reactions with primers for detecting viruses in the test panel. Aliquots of DNA were also examined for the presence of the herpesviruses in the panel. Amplification products were analyzed on a 2% agarose  gel containing ethidium bromide. Routinely, for any virus for which a band corresponding to a positive result is observed, the band is run into low melting agarose, and sequenced directly on the gel slice without further purification. DyeTerminator sequencing was performed on an Applied Biosystems Model 373A automated sequencer . For the TaqMan assay, one-step RT-PCR Ready-Mix Kit (Applied Biosystems) was used. The primers and probe for the quantification of the RNA copy number of WNV used in this study are listed in Virus was isolated by using monolayers of Vero cells grown in tubes. Aliquots of 0.01 and 0.05 mL of whole blood were pretreated with antibiotics (penicillin and streptomycin) for 30 min at room temperature (22°C); 1 mL of Eagle's minimal essential medium containing 2% fetal bovine serum was added to the treated blood samples, and the mixtures were used to inoculate the Vero cell monolayers.-4–10-6) was used to infect fresh Vero cell monolayers. Virus-infected monolayers from the second passage were examined for WNV antigen by immunofluorescence assay (IFA) by using monoclonal antibody H5-46 . This monoclonal immunoglobulin (Ig) M antibody is specific for a glycoprotein epitope on WNV.After incubation for 24 hr at 37°C, the monolayers were rinsed with phosphate-buffered saline, 2 mL of fresh medium was added, and the culture was subsequently monitored daily for cytopathic effects (CPE). Cell monolayers that showed CPE were harvested. To confirm that the infectious agent was WNV, an aliquot of the supernatant serially diluted  in the NS5 region . PCR pro5 region . RT-PCR 5 region) and TaqMan (primers in ENV region) assays; the results are summarized in 6 copies/mL, was found in the blood sample that was collected 3 days after the patient’s neurologic symptoms appeared. WNV genome was also detected in a serum sample collected on day 19 after onset of symptoms. Serologic tests (IgM capture ELISA and IgG ELISA) on serum for Eastern equine encephalitis virus, LACV, POWV, SLEV, and WNV were all negative.To follow up this case, three whole-blood samples and one serum sample were examined by both standard RT-PCR  count 8 mmThe attempt to isolate WNV was carried out in a biosafety level 3 laboratory not routinely used for arbovirus work and located in a building separate from the facility where PCR testing was conducted. This procedure had the dual advantage of minimizing the possibility that any virus recovered originated from a source other than the human specimen and also ensuring that any isolate obtained would not lead to future spurious PCR results. A blood sample collected on September 24, 2001, and a CSF specimen collected on September 26, 2001, were chosen for recovery of the virus because of the presence of high-copy-number viral RNA. On day 6 postinfection, CPE was observed in the tubes inoculated with 0.01 mL and 0.05 mL of blood, but not in the tube inoculated with 0.1 mL of CSF. WNV was confirmed in the second-passage cell cultures by IFA by using WNV-specific monoclonal antibody H5-46.Since only a very small amount of CSF remained after the diagnostic work-up, we carefully designed a protocol to generate PCR bands with sizes ranging from 500 to 1000 bp. This protocol allowed the complete genome sequence of the virus in the CSF to be determined by sequencing overlapping PCR bands (GenBank accession no. AF533540). We used a similar protocol to obtain the genome sequence of the isolate adapted from cell culture and found that the sequence data from the virus in the CSF and from the WNV isolate were identical. A 1648-bp fragment encoding the PreM, M, and part of the 5′-E gene was used for phylogenetic studies . The ana, the patient in this case was elderly and was undergoing treatment for lymphoma. She was unable to mount an immune response as shown by the fact that the results of serologic tests on both CSF and serum specimens were negative.This case is important for several reasons. It represents the first instance in which WNV was recovered from a person in the United States. It is also the first time that the entire genomic sequence of WNV has been obtained from CSF from a human case-patient. The sequence data from the virus directly detected in the CSF and from the WNV isolate from cell cultures are identical. Hindiyeh et al.  reported, because a reduction in immunoglobulins, secondary to impaired B-cell function from rituximab, usually occurs after 3 months of therapy .To our knowledge, all previous attempts to recover WNV from human patients associated with the North American outbreak have been unsuccessful. The ability to recover an infectious isolate in this report may have been contingent on the fact that the patient was immunologically impaired. She had lymphoma and had been undergoing treatment with CHOP plus rituximab for 2 months at the time the WNV infection developed. The relative role of immunosuppression caused by the lymphoma itself compared with the immunosuppression due to treatment of the lymphoma is unclear. At the time of the WNV infection, the patient’s serum IgG levels were moderately suppressed. This condition was most likely the result of lymphoma therapy . In a pr therapy . However therapy . Rituxim therapy . Another, this report is the first of WN encephalopathy in an immunocompromised patient undergoing treatment for lymphoma. The patient’s serologic tests remained negative, and the diagnosis was made by RT-PCR from both CSF and peripheral blood, and by in vitro cultivation of the virus from blood. WNV infection should therefore be considered in the differential diagnosis of patients with lymphoma who exhibit encephalopathy, even if serologic tests are negative for WNV. Extra care should be taken to prevent patients with lymphoma, especially those undergoing treatment, from being exposed to WNV.In summary


Until recently, West Nile (WN) and Kunjin (KUN) viruses were classified as distinct types in the Flavivirus genus. However, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To better define this relationship, we performed sequence analyses on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN isolate from the recent outbreak in New York. Sequence comparisons showed that the KUN virus isolates from Australia were tightly grouped but that the WN virus isolates exhibited substantial divergence and could be differentiated into four distinct groups. KUN virus isolates from Australia were antigenically homologous and distinct from the WN isolates and a Malaysian KUN virus. Our results suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into subgroups on the basis of genetic and antigenic analyses.


Rabies, encephalitis caused by lyssaviruses, was considered universally fatal until a young, unvaccinated patient with bat rabies survived after a new therapeutic approach EurosurveillanceWe report on a patient with rabies due to Duvenhage virus from a bat in Kenya. A very brief description of this case without clinical details and without results of virological and pathological investigations was reported in A bat flew against the face of a 34-year-old Dutch woman, a medical doctor, on a campsite in Tsavo West National Park, Kenya. She swept the animal away and noticed two small superficial bleeding wounds on the right side of the nose. The wounds were washed with water and soap, and cleaned with alcohol swabs. The park wardens and the personnel at the nearby health facility assured her that locally rabies was only known to be carried by dogs and cats. She was not referred for postexposure prophylaxis.Fourteen days after her return to The Netherlands, 23 days after the incident with the bat, she complained of malaise, dizziness, muscle aches, and headache. She had no fever. Two days later, she experienced difficulty with speech and swallowing. There was hypaesthesia of both cheeks and she felt unsteady. She vomited once. She was admitted in isolation the next day, day 4 of complaints. Her past medical history was unremarkable and she did not take medication.Listeria monocytogenes infection, malaria, and trypanosomiasis were considered and ruled out by appropriate tests  and microscopy of blood slides and CSF, PCR for enterovirus, inclusive of enterovirus 71). Neurological considerations included infection by neurotropic viruses, acute disseminated encephalomyelitis, and, initially, anxiety disorder. On day 2 of admission, her temperature was 38.5°C, the dysarthria had worsened, and she complained of difficulty with swallowing. She was agitated and she had a high frequency postural tremor of her hands.On examination she was anxious, but alert and well oriented. Her temperature and other vital parameters were normal. There was no hypersalivation, hydrophobia, or aerophobia, and no difficulty swallowing. The wounds on the nose were resolved; there was no itch or pain. There was a slight dysarthria and she spoke in a staccato way. Apart from the hypaesthesia of both cheeks, further neurological examination was unremarkable. The deep tendon reflexes were normal. On advice of the National Coordination Center for Infectious Diseases (NCCID), she received human anti-rabies immunoglobulin (HRIG), 20 IU/kg im, divided over both quadriceps muscles, and human diploid rabies vaccine (Mérieux) im . Other infections, such as Rift Valley fever, West Nile fever, lymphocytic choriomeningitis, yellow fever, enterovirus 71 and 3 (n<5), no red blood cells, a protein level of 0.23 g/l (n<0.5) and a glucose level of 3.0 mmol/l (blood glucose 4.9 mmol/l). During the following days her dysarthria increased. She complained of weakness of arms and legs and double vision. She developed hypersalivation and became hyperreactive to noises. Her temperature was variable with bouts of fever, up to 39.2°C. On day 6 of admission, the condition acutely worsened; she had an epileptic seizure and aspirated, and oxygenation dropped due to respiratory insufficiency. She was intubated and admitted to the Intensive Care Unit (ICU). The CSF now showed 64 white blood cells  and 25 red blood cells/3 mm3, a protein level of 0.72 g/l, and an elevated IgM index of 0.35. An MRI showed an area of increased signal intensity on T2-weighted images in the posterior part of the medulla oblongata and pons /3 mmand pons . The nexLyssavirus-specific RT-PCR was negative in those specimens. The nuchal biopsy, obtained on day 2 of admission, showed a weak band of correct size on agarose gel in nested RT-PCR in only one of the quadruplicate samples and therefore required confirmation by cloning and sequencing Rabies seemed the probable cause of the encephalitis, but the pharyngo-cervical-brachial variant of Guillain-Barré syndrome (GBS) was also considered. No rabies-specific antibodies had been detected in serum collected before vaccination or in serum or CSF from day 2 of admission, and Clinical samplesSerum, d2 antibody and PCR neg, d7 antibody neg;CSF, d2 antibody and PCR neg, d 6 and d 28 Lyssa PCR neg;Nuchal skin biopsy, d2 PCR pos, see text, IF rabies neg, d10 PCR neg (repeated because of delay with first biopsy);Saliva, d7 Lyssa PCR pos (reported d 13), d10 neg;Cornea smear, d7 IF neg.(Note: results of tests performed.)On day 11 of admission the presence of Duvenhage virus in the nuchal skin biopsy specimen from the 2nd day of admission was confirmed. This may seem like a long delay, but only one of the two agarose gels tested by PCR showed a faint band, and the repeated test in duplicate was negative in both tests. The amplicon had to be cloned as direct sequencing was not successful. Details of virological investigations are presented in Staphylococcus aureus was treated with flucloxacillin iv, 1 g, four times per day for 7 days. On admission to the ICU, the temperature was elevated up to 40.1°C, but after administration of acetaminophen suppositories  the temperature was normal 24 hours later and remained normal thereafter without further measures. Diabetes insipidus, first apparent on day 12 of admission, day 7 of ICU, was regulated with iv fluids to which desmopressin was added on days 13, 16, and 17. Pentobarbital was stopped after 5 days when no bursts were seen on the EEG and midazolam was stopped after 7 days of administration. Twenty-four hours later, 3 days after stopping pentobarbital and while there was no pentobarbital detectable in the serum, the EEG showed a very low voltage background pattern.Blood pressure, heart rhythm, kidney function, and diuresis were regulated by fluids and low dose noradrenaline; a supervening lung infection with MRI abnormalities of the medulla oblongata had increased and now extended to mesencephalon and both dentate nuclei in the cerebellum . Also, tAt postmortem examination no gross abnormalities were found, apart from the pneumonia. The brain was swollen and weighed 1,360 g. Histopathology of the cerebral cortex  showed eThe Platelia Rabies II Kit  was used to detect and titrate IgG anti-rabies virus glycoprotein in serum or plasma. The test was also used to detect specific antibody in CSF.Lyssavirus antigen was detected by the immunofluorescence examination using Bio-Rad Rabies Anti-nucleocapsid Conjugate . Nuchal skin biopsies and corneal smears were examined by the immunofluorescence assay to detect the presence of rabies virus antigen.Cell culture of the various clinical samples was carried out by inoculation of murine neuroblastoma cells and BHK-21 cells propagated in 24-well microtiter plates. CSF was tested without processing, and saliva was made up to a volume of 2 mL with virus transport medium. The other specimens were ground with virus transport medium up to a volume of 5 mL. After centrifugation a volume of 200 µL was added to each well. Immunofluorescence tests with anti-rabies nucleocapside conjugate (Bio-Rad) showed immunopositivity as presented in A rabies-specific diagnostic RT-PCR was performed as previously described A 367–base pair fragment in the nucleoprotein region was sequenced and phylogenetic analysis was performed using Mega 3.1  It should be noted that a Duvenhage strain had not been present in our laboratory.Newborn NIH outbred mice  were inoculated intracerebrally with the various clinical samples . The preparation of the specimens was the same as for the cell culture. The specimens were homogenized and a volume of 15 µL was introduced into the central part of the upper parietal area of the calvaria. The inoculated mice were observed daily for a maximum of 28 days; the inoculated mice did not show symptoms of infection. Rabies virus antigen could not be detected by immunofluorescence of the mouse brains; furthermore, the brains were also negative by PCR.Antibodies against lymphocytic choriomeningitis virus, Rift Valley virus, West Nile virus, and yellow fever virus were determined by immunofluorescence assays or enzyme-linked immunosorbent assays.Tissue was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 6 µm, mounted on organosilane-coated slides , and used for hematoxylin and eosin (H&E) staining. Apart from H&E staining, representative sections of all brain specimens were processed for conventional histochemical staining  and immunohistochemistry.Several blocks of different brain regions  were used for immunohistochemical analysis with neuronal nuclear protein .For the detection of the virus, polyclonal rabbit anti-rabies-nucleoprotein antibody  was used. The immunostaining with this anti-rabies antibody was also performed on material from adrenal gland, salivary gland, pancreas, thyroid, heart, and gastrointestinal tract.2O2 diluted in methanol. Antigen retrieval was performed by incubation for 10 min at 121°C in citrate buffer  or for the anti-RNP with proteinase K pretreatment . After incubation with the primary antibodies overnight at 4°C, ready-for-use Powervision peroxidase system  and 3,3′-diaminobenzidine  or 3-amino-9-ethyl carbazole  were used to visualize the antibodies. Sections were counterstained with haematoxylin. Sections incubated without the primary antibody were essentially blank.Immunohistochemistry was carried out as previously described LyssavirusRabies occurs on all continents apart from Antarctica. The causative viruses belong to the family Rhabdoviridae, genus Diagnosis of rabies depends on demonstration of the virus or antibodies to the virus in various specimens . Several methods are available: RT-PCR, immunofluorescence test, virus neutralization test, and virus isolation in cell culture or animals. In the early stages of disease, results may be negative and tests may have to be performed several times. Once neurological symptoms occur, death is  universally inevitable. Only one patient, who had not received pre- or postexposure prophylaxis, has survived We applied the same methods of coma induction with benzodiazepine and phenobarbital with continuous monitoring of bursts on the EEG and administered ketamine, ribavirin, and amantadine as reported by Willoughby et al. Rabies remains a virtually always fatal disease. In a patient with symptomatic rabies with neutralizing antibodies in the CSF early in the disease, an attempt at intensive treatment inclusive of respiratory and cardiovascular support may be successful. Otherwise, as there are no new data from in vitro and animal studies and no advice from experts on combination treatments, the best possible medical and nursing care with relief of symptoms and comfort care of the patient and support of the family is probably what can be offered at present This is the third human case of rabies due to Duvenhage virus infection. This virus was for the first time isolated in 1970 from a human patient in South Africa This tragic incident is another reminder that even after a minor animal-related injury the wound should be washed thoroughly and postexposure prophylaxis should be given. Where limited access to postexposure prophylaxis may be expected, pre-exposure prophylaxis by vaccination is advised for travellers, especially when travelling with children for longer periods. Human diploid cell vaccine protects against classic rabies virus strains and Duvenhage virus, but not Mokola virus and Lagos bat virus; Duvenhage virus is neutralized by RIG, but Mokola and Lagos bat viruses are not


Results of the first randomized trial comparing on-demand versus planned-relaparotomy strategy in patients with severe peritonitis  indicated no clear differences in primary outcomes. We now report the full economic evaluation for this trial, including detailed methods, nonmedical costs, further differentiated cost calculations, and robustness of different assumptions in sensitivity analyses.An economic evaluation was conducted from a societal perspective alongside a randomized controlled trial in 229 patients with severe secondary peritonitis and an acute physiology and chronic health evaluation (APACHE)-II score ≥11 from two academic and five regional teaching hospitals in the Netherlands. After the index laparotomy, patients were randomly allocated to an on-demand or a planned-relaparotomy strategy. Primary resource-utilization data were used to estimate mean total costs per patient during the index admission and after discharge until 1 year after the index operation. Overall differences in costs between the on-demand relaparotomy strategy and the planned strategy, as well as relative differences across several clinical subgroups, were evaluated.Costs were substantially lower in the on-demand group . Relative differences in mean total costs per patient (approximately 21%) were robust to various alternative assumptions. Planned relaparotomy consistently generated more costs across the whole range of different courses of disease . This difference in costs between the two surgical strategies also did not vary significantly across several clinical subgroups.The reduction in societal costs renders the on-demand strategy a more-efficient relaparotomy strategy in patients with severe peritonitis. These differences were found across the full range of healthcare resources as well as across patients with different courses of disease.ISRCTN51729393 Secondary peritonitis or abdominal sepsis is a serious condition with high in-hospital mortality (estimates vary between 20% and 60%) and considerable major disease-related morbidity -4. PatieThe initial treatment of abdominal sepsis consists of an emergency laparotomy aimed at eliminating the source of the infection. Thereafter, two surgical strategies are used world-wide: planned relaparotomy or relaparotomy on demand. In the planned strategy, a relaparotomy is performed every other day 24 to 36 h) until findings are negative for (ongoing) peritonitis. This strategy may incur the risk of potential surgery-related complications. The on-demand strategy uses 'watchful waiting,' in which a relaparotomy is performed only in those patients showing clinical deterioration or lack of improvement. Fewer relaparotomies are likely to be performed with this strategy [6 h untilThe debate about the preferred relaparotomy strategy (on-demand versus planned) in these patients is longstanding, with both strategies having their proponents. We recently published the results of the first randomized trial comparing these two surgical strategies and demonstrated that patients in the on-demand group did not have a significantly lower rate of adverse clinical outcomes compared with the planned group . HoweverHere we present the economic evaluation comparing costs generated by an on-demand and a planned-relaparotomy strategy from a societal perspective. More details are reported, regarding both methods and the clinical process driving these costs. Sensitivity analyses were performed to evaluate the robustness of the findings for several assumptions and methodologic choices. Furthermore, differences in costs are assessed across patients with different clinical characteristics and courses of disease.This economic evaluation was part of the RELAP trial, a randomized controlled multicenter trial comparing an on-demand relaparotomy strategy with a planned-relaparotomy strategy in patients with severe peritonitis. Details about the design, conduct, and major clinical findings of this trial were reported elsewhere . In brieThe appropriate type of economic evaluation is conditional on the results of the primary end points  and health-related quality-of-life (HR-QoL). In case of one clearly superior strategy, a cost-effectiveness analysis (CEA) would be required to combine clinical and economic outcomes. In case of comparable outcomes in clinical effectiveness, a cost-minimization analysis (CMA) would suffice . As clinHealthcare utilization and other resources were prospectively documented for individual patients , by usinThe cost analysis was set up from a societal perspective, which consists of three cost categories . These iTo exclude potential protocol-driven costs, we assessed the extent to which procedures and follow-up visits are part of the usual care for these patients or are relevant only for the conduct of the trial. Basically, some surgical and diagnostic interventions formed protocols for the study, but this was because they are inherent to the surgical strategy; other procedures and interventions were at the discretion of the surgeon, reflecting usual care.Data on resource utilization during the index hospital admission included the number of surgical interventions (including relaparotomies), percutaneous drainage procedures, diagnostic procedures , ultrasound (US), plain radiograph of the abdomen), postoperative hospital stay from index laparotomy onward, and ICU stay. The initial summary cost analyses reported with the main clinical findings  were furAll data were registered alongside the clinical study. Resource utilization after discharge was documented in specifically developed, self-administered questionnaires that were sent to surviving discharged patients at 3, 6, 9, and 12 months of follow-up. Patients reported use of primary, secondary, and paramedic healthcare services. Employment status and absence from paid work was also documented by using the Health and Labour Questionnaire . NonrespEstimates of unit costs were derived from different sources [see Additional File Costs were calculated for individual patients by multiplying actually used healthcare resources and unit costs. Data concerning postdischarge healthcare utilization were not always complete because of partial or nonresponse to the self-administered questionnaires. In these cases, the average resource utilization for an out-of-hospital day was estimated within each study/treatment group, and extrapolated over the total out-of-hospital period. Productivity costs due to illness or recovery in patients younger than 65 years were estimated based on patient-reported absences from paid work. Productivity costs were calculated by using a friction cost approach, which assumes that after a friction period (154 days), each employee is replaced in the workforce .All analyses were performed according to the intention-to-treat principle . Data maMean volumes of resource utilization and associated costs during the index admission and 1-year follow-up were estimated for the two surgical strategies. Differences in volumes were tested for significance by using a nonparametric Wilcoxon Mann-Whitney test.Total costs per patient were estimated as the sum of direct medical costs, direct nonmedical costs, and indirect costs. Differences in total costs between the two surgical strategies were tested based on their geometric means .Robustness of our results were evaluated in sensitivity analyses regarding the extent to which cost differences can be attributed to costs of relaparotomies alone, as well as for unit costs for a range of cost drivers ,6. We evFinally, we hypothesized that the relative difference between the on-demand relaparotomy strategy and the planned strategy could be different across clinical subgroups: between patients with and without any major comorbidity , patients with high (>20) versus patients with lower (11-20) APACHE-II scores, and between patients surviving and those who died before 12 months of follow-up. A linear regression model was used, with the log-transformed costs as the outcome measure to improve the normality of the residuals required in such models. Differences between clinical subgroups were estimated and statistically tested by adding this as an interaction effect with the type of surgical strategy to the model.In total, 229 patients were correctly randomized and included in this study. In both surgical strategies, one patient withdrew informed consent, and one patient was lost to follow-up, meaning that data on the initial admission were available for 229 patients (114 on-demand and 115 planned strategy), and data on the 1-year follow-up, for 225 patients (112 on-demand and 113 planned strategy).Demographic and clinical baseline characteristics of these randomized patients are presented in Table Data available for analysis from the index admission and follow-up admissions were available for all patients. Additional data on use of healthcare resources outside the hospital , travel, and absence from work were reported for at least one follow-up period by 76 patients in the on-demand group and 74 patients in the planned group. For both strategies, mean costs per out-of-hospital day were estimated and extrapolated to patients who did not report this part of the follow-up.P < 0.001). Although in the on-demand group, significantly fewer relaparotomies comprised additional surgical procedures , the associated mean costs per patient  of these additional procedure differed only marginally (€332).Results of the cost analyses are presented by reporting mean volumes, total costs, mean costs per patient per strategy for the on-demand group and the planned group for resource utilization during the index admission, and follow-up Table . Mean coP = 0.038)) for the planned group) did not compensate for the lower numbers of relaparotomies in the on-demand group.Higher costs generated by additional US- or CT-guided percutaneous drainages (€147 (27% received PCD) for the on-demand group and €233  for the planned-relaparotomy group . Costs associated with mechanical ventilation showed a similar picture, with mean costs estimated as €3,080  for the on-demand group and €4,360  for the planned group . Costs generated by hospital stay on the ward (excluding ICU days) were estimated as €11,609 for the on-demand group and €11,784 for the planned-relaparotomy group. Although the total length of hospital stay for the index admission was substantially shorter for patients in the on-demand group , length of stay on the ward was comparable .As a large majority of patients were admitted to the ICU, substantial costs were generated by the ICU stay. Mean estimated costs per patient generated by the ICU stay were €21,040  in the on-demand group and €31,248 . Mean costs per patient for medication were €474 versus €619; for blood products, €89 versus €178; and for enterostomy care, €741 and €917, comparing the on-demand group and the planned-relaparotomy group. Direct medical costs during admission were significantly lower in the on-demand group, with a mean difference of €14,418 .P = 0.88), with mean costs €4,280 for the on-demand patients versus €5,083 for the planned patients). On average, enterostomy care and associated costs during follow-up were considerable. Patients had an enterostomy for a mean of 140 days  in the on-demand group and 150 days  in the planned-relaparotomy group (P = 0.44).Costs associated with readmissions during the 1-year follow-up were comparable  and had shorter stays in rehabilitation centers than did the planned-relaparotomy group . Differences between the study groups in utilization and associated costs of outpatient care and visits to secondary healthcare providers  were marginal. The direct medical costs during follow-up (between discharge and 12 months after the index laparotomy) were lower in the on-demand group, with a mean difference per patient of €4,069 .Distinct differences in resource utilization and associated costs of outpatient and secondary healthcare providers were found for home care supplied by district nurses and stay in rehabilitation centers, in which the on-demand group received less home care . Costs associated with lost productivity for these days were estimated as €2,854 and €2,048, respectively.Among patients reporting to have paid work, only the occasional patient reported a return to work within 12 months. For all nonresponding patients younger than 65 years, we therefore assumed that they were at least absent for the full friction period (154 days). The mean number of days absent from paid work was estimated as 70 days in the on-demand group versus 50 days in the planned group . Of these total costs, 75% were generated during the initial admission, of which 45% to 55% were ICU costs.Overall, for the 1-year study period, mean total costs per patient associated with the on-demand strategy were €65,768 versus €83,450 with the planned strategy , whereas costs associated with both strategies appear to be rather comparable in others  Table . In patiWe present an economic evaluation within a randomized clinical trial comparing two commonly used surgical strategies for patients with secondary peritonitis after their initial emergency laparotomy, on-demand relaparotomy and planned relaparotomy. In an earlier publication focusing on the clinical outcomes of the RELAP trial, we demonstrated that patients in the on-demand group did not have a significantly lower rate of poor outcomes compared with the planned group . The resThe observed cost differences were predominantly related to lengthier ICU stays and duration of mechanical ventilation during the index admission period. Costs of rehabilitation centers and home care and of readmissions to a general hospital during follow-up were also substantial contributors to these cost differences. Although the planned strategy per definition involved at least one relaparotomy procedure, costs generated only by this extra procedure were only a mere fraction of the encountered cost differences. When costs associated with relaparotomy procedures were disregarded, major cost differences between the surgical strategies remained present.An important component of the total direct medical costs was the ICU stay . Consequently, total costs were highly influenced by the unit costs estimate for an ICU day. We used a reference price based on data from a range of general and academic hospitals in the Netherlands . In the In general, resource utilization was found to be higher in the planned group than in the on-demand group. Therefore, adjustments in unit costs would result in changing total costs, rather than affecting the difference between on-demand and planned relaparotomy. Total costs varied to some degree with the different assumptions regarding unit-cost prices, but the relative difference between the strategies remained consistent across these analyses. On average, the on-demand strategy generated approximately 21% less costs than planned relaparotomy. Per 1,000 patients admitted to an emergency room with severe peritonitis, half of whom are currently operated on according to the planned strategy, some €10 million could be saved.No other studies have reported a detailed description of costs associated with resource utilization generated by abdominal sepsis patients treated by either strategy, and we can compare volumes only; our findings were comparable to those of a retrospective study comparing on-demand and planned relaparotomy, reporting similar figures for average length of hospital stay (49.5 vs. 52.0 (including ICU) and average length of ICU stay (12.6 versus 17.8 days). Duration of mechanical ventilation was a few days longer (10.3 versus 13.9) as compared with our results (8.5 versus 12 days), but the difference  was consistent with our findings [This study has some strengths and limitations. First, the economic evaluation was performed as part of a randomized, controlled trial that stratified for severity of disease, ensuring that the patients in both strategies were comparable with respect to clinical and prognostic factors. Differences in resource utilization and related costs can therefore confidently be attributed to the surgical strategy. Furthermore, the economic evaluation was based on data on resource utilization required for the clinical trial and extended with additional relevant information acquired with self-administered questionnaires. This bottom-up strategy provided insight into the healthcare process and main cost-driving factors. Although the majority of costs were generated during index admission, systematic documentation during follow-up demonstrated that these differences remain unchanged from a societal perspective.Currently, support of the on-demand strategy is growing -27, and This economic evaluation prospectively demonstrated that resource utilization and associated costs generated during treatment and follow-up of severe peritonitis were substantially lower for an on-demand strategy compared with a planned strategy. These differences were found across the full range of healthcare resources as well as across patients with different courses of disease. Considering that patients in the on-demand relaparotomy strategy group had a lower  rate of adverse outcomes compared with the planned-relaparotomy group , the red• Patients with severe peritonitis require intensive monitoring and medical treatment, often including lengthy ICU stays, and therefore incur substantial costs to the healthcare system.• The first randomized clinical trial comparing on-demand versus a planned-relaparotomy strategy in patients with severe peritonitis  indicated no clear differences in primary clinical outcomes.• To assess the economic impact of differences in resource use, we performed a full economic evaluation from a societal perspective alongside this trial.• Mean total costs per patient were 20% lower in the on-demand group as compared with the planned group.• The substantial difference in costs renders the on-demand strategy a far more efficient relaparotomy strategy in patients with severe peritonitis.APACHE: Acute Physiology and Chronic Health Evaluation; CRF: clinical report form; CEA: cost-effectiveness analysis; CMA: cost-minimization analysis; CT: computed tomography; FFP: fresh frozen plasma; HR-QoL: health-related quality of life; ICU: intensive care unit; OD: on demand relaparotomy; PCD: percutaneous drainage; PR: planned relaparotomy; RELAP trial: randomized controlled trial comparing relaparotomy on demand with planned relaparotomy; US: ultrasound; US$: United States dollar; 95% CI: 95% confidence interval.The authors declare that they have no competing interests.MAB, DJG, HO, JBR, and CAJMB designed the clinical study. BCO, CAJMB, and MAB designed the economic evaluation alongside the clinical trial. OR and CWM were responsible for the coordination of the study, including contacting patients and collecting and entering data. KRB and OR were responsible for assessment and processing of follow-up data. BCO, KRB, OR, JBR, and MAB were responsible for the cost analyses. HGG, PWG, BL, MFG, and EPS were responsible for including more than 10% of the randomized patients in their participating hospital. BCO analyzed data and prepared initial versions of the manuscript. BCO, KRB, OR, JBR, and MAB were responsible for the final manuscript. BCO, KRB, OR, JBR, and MAB interpreted and discussed all data. All authors read, reviewed, and approved the final manuscript.Table reporting units of resource use, unit costs (€), valuation method and volume source used for the cost analyses.Click here for file


Cryptosporidium hominis, C. parvum, C. canis, and C. felis in 25, 7, 1, and 1 HIV-positive persons from Jamaica, respectively; 1 person had both C. hominis and C. felis. Multilocus sequence typing indicated the presence of a homogeneous but geographically distinct C. hominis population in Jamaica.A cryptosporidiosis survey showed the presence of  Cryptosporidium species and subtypes ; Cryptosporidium spp. were identified on the basis of PCR–restriction fragment length polymorphism analysis of the small subunit ribosomal RNA gene as described (C. hominis and C. parvum subtypes was based on sequence polymorphism at the 60-kDa glycoprotein gene (GP60) locus by using the nomenclature described , a serine repeat antigen (MSC6–7 minisatellite), a hypothetical retinitis pigmentosa GTPase regulator (RPGR minisatellite), and a hydroxyproline-rich glycoprotein (DZHRGP minisatellite and microsatellite) in chromosome 6 and the 70-kDa heat shock protein (HSP70 minisatellite) in chromosome 2. Nomenclature and classification of subtypes at these 5 loci have been described  C. hominis specimens had subtype IbA10G2; 3 had subtype IeA12G3T3. All 7 C. parvum specimens belonged to subtype IIcA5G3d, which was identical to C. parvum identified in children in South Africa (GenBank accession no. AF440636).Genotype analysis of the 35 C. hominis. At the CP47 locus, all 19 specimens analyzed belonged to subtype IA39G22. Most of the other loci were also monomorphic, and subtypes at these loci were identified by amplicon sizes. All specimens were 590 bp at the DZHRGP locus, 358 bp at the RPGR locus, and 1,095 bp at the HSP70 locus. At the MSC6–7 locus, 18 of the 19 C. hominis specimens analyzed were 494 bp; 1 was 509 bp.Results of the MLST analysis of the additional 5 loci showed limited gene diversity in C. parvum, although all specimens belonged to IIcA5G3d subtype at the GP60 locus. There were 3 subtypes at the DZHRGP locus. Subtype 1 was 496 bp and was found in 4 specimens. Subtype 2 was also 496 bp but had 1 single nucleotide polymorphism at position 314; this subtype was seen in 2 specimens. Subtype 3 was 499 bp and was found in 1 specimen. The HSP70 locus had 2 subtypes, differentiated by the presence of a single nucleotide polymorphism (a change of C to T at position 172), with 6 of the 7 specimens in 1 subtype. All C. parvum specimens were monomorphic at the CP47, MSC6–7, and RPGR loci. Results of the C. hominis and C. parvum subtyping at the 5 loci are shown in the Diversity was more pronounced in the MLST analysis of C. hominis and C. parvum, an MLST analysis was conducted to compare MLST types from Jamaica with those from other geographic areas of similar GP60 subtype families Ib, Ie and IIc . All sequences for each specimen at the 5 loci were concatenated and multilocus subtypes generated. The analysis showed C. hominis multilocus subtypes from Jamaica clustered in 1 group, regardless of their GP60 subtype designation. Specimens of the same GP60 subtypes from other regions clustered in clades separate from the Jamaica specimens, forming largely distinct monophyletic groups defined by geographic origin. Specimens from Kenya and India clustered in 1 clade because of extensive human migration between the 2 countries. Although all C. parvum specimens from Jamaica were identified as IIcA5G3d, 4 MLST types were present, highlighting the extent of genetic diversity of C. parvum in this study. The relationship of the multilocus subtypes based on the 5 loci inferred by neighbor-joining analysis is shown in the To measure the degree of heterogeneity of C. hominis and anthroponotic C. parvum (the IIc subtype family) in 33 (94%) of 35 specimens analyzed. Because all C. parvum–positive specimens belonged to the anthroponotic subtype family IIc and because 1 of the C. felis–infected patients had concurrent infection with C. hominis, it is possible that some of the C. canis and C. felis infections seen in this study were transmitted through anthroponotic pathways.Results of our study show that anthroponotic transmission is important in the epidemiology of cryptosporidiosis in Jamaica. This conclusion is based on our finding of C. hominis subtype families (Ib and Ie); most belonged to the IbA10G2 subtype. Although the sample size is small, homogeneity within C. hominis at the GP60 locus is unusual, as common subtype families such as Ia and Id are usually equally abundant in most developing countries , belonged mostly to 1 MLST group distinct from other subtypes from other geographic regions. These results suggest that a unique C. hominis parasite population is being transmitted in Jamaica. Heterogeneity was higher in the C. parvum population despite the smaller number of specimens that we analyzed. As with C. hominis, a unique population of C. parvum in Jamaica also seems to exist. These results further show the distinct population of Cryptosporidium spp. that can arise because of geographic segregation. When one considers that all C. parvum specimens belonged to only 1 GP60 subtype, whether the high diversity seen with the MLST is due to recent expansion of an ancestral type in the region is not clear. A study of population genetics of Cryptosporidium spp. from humans in Haiti showed limited multilocus subtypes in both C. hominis and C. parvum, which was interpreted as an indication for an epidemic clonal population for both species (All C. hominis and C. parvum populations in Jamaica that can be differentiated from other geographic regions, including the nearby United States, is notable and requires further investigations. The distinction is important in mapping the transmission of the parasite, especially where tracking infection pathways is necessary, such as in investigations of outbreaks and traveler’s diarrhea. The fact that C. hominis from different areas may have an identical GP60 subtype but a different MLST subtype is also noteworthy, as some C. hominis subtypes, such as IbA10G2, are commonly associated with water-borne outbreaks in industrialized countries. The clinical manifestations of the GP60 subtypes in relation to the distinct MLST from different geographic areas needs to be studied further. This would clarify how geographic segregation in Cryptosporidium MLST subtypes relates to GP60 subtypes that have various pathogenicities.The occurrence of unique multilocus subtypes in both