Spleen Organoid Units Generate Functional Human and Mouse Tissue-Engineered Spleen in a Murine Model.

Introduction: Splenectomy is common after trauma or hematologic disease, and alters immune protection against pathogens, which may lead to fulminant infection with high mortality. Yet the spleen has demonstrable regenerative capacity and cells might be recovered and reimplanted at the time of injury or excision to avoid these risks. Methods: Tissue-engineered spleen (TESp) was generated from ActinGFP mice (mTESp) or human donor spleen (hTESp) through implantation of spleen organoid units (spleen OU), in NOD/SCID mice with concurrent splenectomy, on a biodegradable scaffold. Explants were evaluated and blood smears were obtained to investigate the presence of target cells or Howell-Jolly bodies, which are erythrocyte sequelae of asplenia. Results: TESp was generated from mouse (mTESp) and human (hTESp) donor cells with essential splenic components: red and white pulp with trabeculae. mTESp and hTESp demonstrated green fluorescent protein- or lamin-positive costaining with proliferating cell nuclear antigen, CD4, and CD11c, identifying proliferative donor cells and key immune components of the spleen of donor origin. Animals with hTESp and mTESP combined with splenectomy had significantly fewer Howell-Jolly bodies on blood smears than controls. Conclusion: TESp from mouse and human donor cells can be generated by 4 weeks and contains donor immune cells identified by CD4 and CD11c. TESp reduces postsplenectomy erythrocyte inclusions, indicating possible function. Impact Statement Overwhelming postsplenectomy infection is rare but highly mortal. Tissue-engineered spleen (TESp) was generated from murine (mTESp) and human (hTESp) donors and appeared histologically similar to native spleen. Both mTESp and hTESp demonstrated proliferative cells of donor spleen origin. Importantly, functional cells were demonstrated on imaging with a corresponding reduction in the number of erythrocyte inclusions in blood smears that are typically identified in patients with asplenia and indicate a lack of clearance by functional spleen tissue. Taken together, these findings indicate that this approach might be clinically relevant as a future human therapy.

Screening for blunt cerebrovascular injuries in pediatric trauma patients.

BACKGROUND: Adult imaging for blunt cerebrovascular injuries (BCVI) is based on the Denver and Memphis screening criteria where CT angiogram (CTA) is performed for any one of the criteria being positive. These guidelines have been extrapolated to the pediatric population. We hypothesize that the current adult criteria applied to pediatrics lead to unnecessary CTA in pediatric trauma patients. STUDY DESIGN: At our center, a 9-year retrospective study revealed that strict adherence to the Denver and Memphis criteria would have resulted in 332 unnecessary CTAs out of 2795 trauma patients with only 0.3% positive for BCVI. We also conducted a retrospective chart review of 776,355 pediatric trauma patients in the National Trauma Data Bank (NTDB) from 2007 to 2014. Data collection included children between ages 0 and 18, ICD-9 search for blunt cerebrovascular injury, and ICD-9 codes that applied to both Denver and Memphis criteria. RESULTS: Of 776,355 pediatric trauma activations, 81,294 pediatric patients in the NTDB fit the Denver/Memphis criteria for screening CTA neck or angiography based on ICD-9 codes, while only 2136 patients suffered BCVI. Strict utilization of the Denver/Memphis criteria would have led to a negative CTA in 79,158 (97.4%) patients. Multivariate regression analysis indicates that patients with skull base fracture, cervical spine fractures, cervical spine fracture with cervical cord injury, traumatic jugular venous injury, and cranial nerve injury should be considered part of the screening criteria for BCVI. CONCLUSION: Our study suggests the Denver and Memphis criteria are inadequate screening criteria for CTA looking for BCVI in the pediatric blunt trauma population. New criteria are needed to adequately indicate the need for CT angiography in the pediatric trauma population. LEVEL OF EVIDENCE: IV.

Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish.

Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/ß-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/ß-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/ß-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.

Non-Operative Management is More Cost-Effective than Immediate Operation in Perforated Appendicitis Patients with Seven or More Days of Symptoms.

BACKGROUND: Management of perforated appendicitis in children remains controversial. Nonoperative (NO) and immediate operative (IO) strategies are used with varying outcomes. We hypothesized that IO intervention for patients with perforated appendicitis would be more cost-effective than NO management. METHODS: A retrospective chart review of all patients with appendicitis from 2012 to 2015 was performed. Patients with perforated appendicitis were defined by evidence of perforation on imaging. We excluded patients who presented with sepsis, organ failure, and ventriculoperitoneal shunts. NO management was determined by surgeon preference. Univariate and multivariate analyses were performed. RESULTS: IO was performed on 145 patients with perforated appendicitis, whereas 83 were treated with NO management. Compared to IO patients, NO patients incurred higher overall costs, greater length of stay, more readmissions, complications, peripherally inserted central venous catheter lines, interventional radiology drains, and unplanned clinic and emergency department visits (P < 0.0001 for all). Multivariate analysis adjusting for age, days of symptoms, admission C-reactive protein and white blood cell count revealed that NO management was independently associated with increased costs (OR 1.35, 1.12-1.62, 95% CI). Cost curves demonstrated that total cost for IO surpasses that of NO management when patients present with greater than 6.3 d of symptoms (P = 0.01). CONCLUSIONS: Our data suggest that IO is more cost-effective than NO management for patients with perforated appendicitis who present with less than 6.3 d of symptoms, after which point, NO management is more cost-effective. LEVEL OF EVIDENCE: IV.

Is less more? Laparoscopic versus open Ladd's procedure in children with malrotation.

BACKGROUND: With the advent of minimally invasive techniques, laparoscopic Ladd's procedure is increasingly used to treat children with malrotation, yet evidence regarding its safety and efficacy is lacking. We hypothesize that operative and postoperative outcomes with the open technique are superior to the laparoscopic Ladd's procedure. METHODS: We conducted a 5-y retrospective chart review of all patients who underwent Ladd's procedure at our institution from 2010-2015. Exclusion of patients included those with concomitant conditions, such as poor gut perfusion, significant reflux, tracheoesophageal fistula, failure to thrive requiring concomitant gastrostomy, and biliary atresia. Kruskal-Wallis and Mann-Whitney tests were used where appropriate. RESULTS: Between 2010 and 2015, of 130 patients who underwent Ladd's procedure, 77 met inclusion criteria. Sixty-two patients underwent initial open surgery, 15 patients underwent laparoscopy, seven of which were converted to open. Patients undergoing open surgery were younger compared to the laparoscopic groups. Thirty-three of the 77 malrotation patients (43%) presented with volvulus, 27 underwent open surgery, four had laparoscopic converted to open procedures, and two patients underwent laparoscopic Ladd's without incident. Laparoscopy resulted in increased operative time and clinic visits. Patients undergoing laparoscopic to open surgery had longer operative times, time to resume diet, and length of hospital stay. No difference was noted in complications among the groups. CONCLUSIONS: Although minimally invasive approaches are becoming increasingly used, no evidence supports laparoscopic superiority over open Ladd's procedure. We found that open surgery was associated with shorter operating times and fewer clinic visits. Furthermore, laparotomy remains the favored procedure for patients presenting with volvulus.

Evolving understanding of neonatal necrotizing enterocolitis.

PURPOSE OF REVIEW: Necrotizing enterocolitis (NEC) is a devastating disease that predominately affects premature neonates. The pathogenesis of NEC is multifactorial and poorly understood. Risk factors include low birth weight, formula-feeding, hypoxic/ischemic insults, and microbial dysbiosis. This review focuses on our current understanding of the diagnosis, management, and pathogenesis of NEC. RECENT FINDINGS: Recent findings identify specific mucosal cell types as potential therapeutic targets in NEC. Despite a broadly accepted view that bacterial colonization plays a key role in NEC, characteristics of bacterial populations associated with this disease remain elusive. The use of probiotics such as lactobacilli and bifidobacteria has been studied in numerous trials, but there is a lack of consensus regarding specific strains and dosing. Although growth factors found in breast milk such as epidermal growth factor and heparin-binding epidermal growth factor may be useful in disease prevention, developing new therapeutic interventions in NEC critically depends on better understanding of its pathogenesis. SUMMARY: NEC is a leading cause of morbidity and mortality in premature neonates. Recent data confirm that growth factors and certain bacteria may offer protection against NEC. Further studies are needed to better understand the complex pathogenesis of NEC.

Hospital admission unnecessary for successful uncomplicated radiographic reduction of pediatric intussusception.

PURPOSE: After radiologic reduction, patients with ileocolic intussusception are often admitted. We hypothesize that discharge of stable patients after 4 h of emergency department (ED) observation does not result in an increase of adverse outcomes. METHODS: We retrospectively reviewed pediatric patients with ileocolic intussusception between 2011 and 2016, managed with either 24-h inpatient or 4-h ED observation. Outcomes included length of stay, adverse outcomes, and total hospital charges. RESULTS: Fifty-one patients were managed with ED observation and 79 with inpatient observation. Recurrence rates, time to recurrence, and adverse outcomes were similar in both protocols. Total recurrence rates for ED observation was 15% versus 14% for inpatient observation. ED observation reduced time in the hospital by 26.8 h (4.9 versus 31.7 h). CONCLUSION: Discharging patients following uncomplicated hydrostatic reduction of ileocolic intussusception after a 4-h observation period does not result in an increase in adverse outcomes.

Host conditioning and rejection monitoring in hepatocyte transplantation in humans.

BACKGROUND & AIMS: Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Here we aimed to use preparative liver-directed radiation therapy, and continuous monitoring for possible rejection in an attempt to overcome these limitations. METHODS: Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression. RESULTS: Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67+ hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343±48µM (normal 30-119µM) to 854±25µM (treatment goal ≤360µM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900µM during supervised follow-up, but graft loss occurred after follow-up became inconsistent. CONCLUSIONS: Radiation preconditioning and serial rejection risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure. LAY SUMMARY: Hepatocyte transplantation can potentially be used to treat genetic liver disorders but its application in clinical practice has been impeded by inefficient hepatocyte engraftment and the inability to monitor rejection of transplanted liver cells. In this study, we first show in non-human primates that pretreatment of the host liver with radiation improves the engraftment of transplanted liver cells. We then used this knowledge in a series of clinical hepatocyte transplants in patients with genetic liver disorders to show that radiation pretreatment and rejection risk monitoring are safe and, if optimized, could improve engraftment and long-term survival of transplanted hepatocytes in patients.

Hematopoietic chimerism following allotransplantation of the spleen, splenocytes or kidney in pigs.

BACKGROUND: Mixed chimerism is associated with donor-specific tolerance. Spleen or splenocyte allotransplantation (Tx) is recognized as potentially tolerogenic. There is no definitive report comparing chimerism levels following spleen and splenocyte Tx in a large animal model. We have compared chimerism after spleen, splenocyte, or kidney Tx in pigs. METHODS: Outbred (n = 5) and MHC-defined miniature (n = 1) pigs underwent orthotopic spleen Tx. Outbred pigs received splenocytes through a systemic vein (n = 1) or the portal vein (n = 3). Kidney Tx (n = 2) or concomitant Tx of spleen+kidney (n = 2) was carried out. All except one recipient pigs were irradiated (700 cGy thymic and 100-125 cGy whole body) on day-2. Cyclosporine or tacrolimus was administered for 42 days. All donors were males and all recipients were females; chimerism in the blood was determined by Quantification-PCR for the donor Y chromosome. Mixed lymphocyte reaction (MLR) was performed before and after Tx. RESULTS: One week after spleen Tx in outbred and MHC-defined pigs, chimerism ranged between 0.8 and 22.5%, and 5.4-20.1%, respectively, and remained between 17.7 and 67.4%, and 2.2-7.4%, respectively, until day 28. One week after splenocyte Tx, chimerism ranged between 0.1 and 8.5%, and decreased to 0.1-0.8% at 3-4 weeks. There was no detectable chimerism 14 days after kidney Tx. The response on MLR of all recipient pigs to donor cells was decreased after Tx, except in one case of splenocyte Tx, indicating that this pig might have become sensitized. After discontinuation of immunosuppression, most isolated spleen or kidney grafts were not rejected, but the kidney was rejected after concomitant spleen+kidney Tx. CONCLUSIONS: There was a significantly higher level of blood chimerism following spleen Tx compared to splenocyte or kidney Tx. However, concomitant Tx of spleen+kidney may be associated with accelerated kidney graft rejection.

A nonhuman primate model of human radiation-induced venocclusive liver disease and hepatocyte injury.

BACKGROUND: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. METHODS AND MATERIALS: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. RESULTS: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. CONCLUSIONS: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.

Testicular recovery after irradiation differs in prepubertal and pubertal non-human primates, and can be enhanced by autologous germ cell transplantation.

BACKGROUND: Although infertility is a serious concern in survivors of pediatric cancers, little is known about the influence of the degree of sexual maturation at the time of irradiation on spermatogenic recovery after treatment. Thus, we address this question in a non-human primate model, the rhesus monkey (Macaca mulatta). METHODS: Two pubertal (testis size 3 and 6.5 ml, no sperm in ejaculate) and four prepubertal (testis size 1 ml, no sperm in ejaculate) macaques were submitted to a single fraction of testicular irradiation (10 Gy). Unilateral autologous transfer of cryopreserved testis cells was performed 2 months after irradiation. Testicular volume, histology and semen parameters were analyzed to assess irradiation effects and testicular recovery. RESULTS: Irradiation provoked acute testis involution only in the two pubertal monkeys. Subsequently, testis sizes recovered and sperm was present in the ejaculates. Longitudinal outgrowth of seminiferous tubules continued, and, in testes without autologous cell transfer, 4-22% of tubular cross sections showed spermatogenesis 2 years after irradiation. In contrast, the four prepubertal monkeys showed neither a detectable involution as direct response to irradiation, nor a detectable growth of seminiferous tubules later. However, two of these animals showed spermarche 2 years after irradiation, and 8-12% of tubules presented spermatogenesis. One prepubertally irradiated monkey presented fast growth of one testis after cell transfer, and showed spermarche 1 year after irradiation. The infused testis had spermatogenesis in 70% of the tubules. The contralateral testis remained smaller. CONCLUSION: We conclude that irradiation before puberty has a severe detrimental effect on outgrowth of seminiferous tubules. But, within the seminiferous epithelium, spermatogenetic recovery occurs at a low rate with no detectable relation to the maturity of the epithelium at irradiation. We also show that autologous testis cell transplantation can enhance spermatogenesis, but only in isolated cases.

Attempted depletion of passenger leukocytes by irradiation in pigs.

Allograft/xenograft rejection is associated with "passenger leukocyte" migration from the organ into recipient lymph nodes. In Study 1, we attempted to deplete leukocytes from potential kidney "donor" pigs, using two regimens of total body irradiation. A dose of 700 cGy was administered, followed by either 800 cGy ("low-dose") or 1,300 cGy ("high dose") with the kidneys shielded. Neither regimen was entirely successful in depleting all leukocytes, although remaining T and 8 cell numbers were negligible. Study 2 was aimed at providing an indication of whether near-complete depletion of leukocytes had any major impact on kidney allograft survival. In non-immunosuppressed recipient pigs, survival of a kidney from a donor that received high-dose irradiation was compared with that of a kidney taken from a non-irradiated donor. Kidney graft survival was 9 and 7 days, respectively, suggesting that depletion had little impact on graft survival. The lack of effect may have been related to (i) inadequate depletion of passenger leukocytes, thus not preventing a direct T cell response, (ii) the presence of dead or dying leukocytes (antigens), thus not preventing an indirect T cell response, or (iii) constitutive expression of MHC class II and B7 molecules on the porcine vascular endothelium, activating recipient T cells.

Measurement of relative output factors for the 8 and 4 mm collimators of Leksell Gamma Knife Perfexion by film dosimetry.

Three types of films, Kodak EDR2, Gafchromic EBT, and Gafchromic MD-V2-55, were used to measure relative output factors of 4 and 8 mm collimators of the Leksell Gamma Knife Perfexion. The optical density to dose calibration curve for each of the film types was obtained by exposing the films to a range of known doses. Ten data points were acquired for each of the calibration curves in the dose ranges from 0 to 4 Gy, 0 to 8 Gy, and 0 to 80 Gy for Kodak EDR2, Gafchromic EBT, and Gafchromic MD-V2-55 films, respectively. For the measurement of relative output factors, five films of each film type were exposed to a known dose. All films were scanned using EPSON EXPRESSION 10000 XL scanner with 200 dpi resolution in 16 bit gray scale for EDR2 film and 48 bit color scale for Gafchromic films. The scanned images were imported in the red channel for both Gafchromic films. The background corrections from an unexposed film were applied to all films. The output factors obtained from film measurements were in a close agreement both with the Monte Carlo calculated values of 0.924 and 0.805 for 8 and 4 mm collimators, respectively. These values are provided by the vendor and used as default values in the vendor's treatment planning system. The largest differences were noted for the Kodak EDR 2 films (-2.1% and -4.5% for 8 and 4 mm collimators, respectively). The best agreement observed was for EBT Gafchromic film (-0.8% and +0.6% differences for 8 and 4 mm collimators, respectively). Based on the present values, no changes in the default relative output factor values were made in the treatment planning system.

Unintended attenuation in the Leksell Gamma Knife Perfexion calibration-phantom adaptor and its effect on dose calibration.

The calibration of Leksell Gamma Knife Perfexion (LGK PFX) is performed using a spherical polystyrene phantom 160 mm in diameter, which is provided by the manufacturer. This is the same phantom that has been used with LGK models U, B, C, and 4C. The polystyrene phantom is held in irradiation position by an aluminum adaptor, which has stainless steel side-fixation screws. The phantom adaptor partially attenuates the beams from sectors 3 and 7 by 3.2% and 4.6%, respectively. This unintended attenuation introduces a systematic error in dose calibration. The overall effect of phantom-adaptor attenuation on output calibration of the LGK PFX unit is to underestimate output by about 1.0%.

Dosimetric comparison of the Leksell Gamma Knife Perfexion and 4C.

OBJECT: The recently introduced Leksell Gamma Knife (LGK) Perfexion is an entirely new system with a different beam geometry compared with the LGK 4C. The new Perfexion system has 192 cobalt-60 sources that are fixed on 8 sectors (each sector has 24 sources). Each sector can be moved independently of the others and can be set to 1 of 5 different positions: 3 positions defining collimator sizes of 4, 8, and 16 mm; an off position (sources are blocked); and a home position. The purpose of this study is to compare the dosimetric characteristics of the GK 4C and the Perfexion models. This comparison is important especially for the treatment of functional disorders when only a single shot with the 4- or 8-mm collimator is used. METHODS: A 160-mm-diameter spherical polystyrene phantom was used for all measurements and calculations. The irradiation geometry consisted of the placement of a single shot at the center of this phantom. Comparisons were made among different dosimetric parameters obtained from calculations performed using Leksell GammaPlan v. 8.0 and measurements performed using film dosimetry. The dosimetric parameters investigated were dose profiles for all collimators in all 3 stereotactic planes (x, y, and z) including the full width at half maximum and the penumbra for each profile, cumulative dose-volume histograms, the volume encompassed by the 50% isodose surface, the mean doses delivered to a defined matrix volume, and relative output factors for all collimator sizes. RESULTS: There was excellent agreement between the dosimetric parameters of GK 4C and Perfexion for the 4- and 8-mm collimators. CONCLUSIONS: The results of this study suggest that consistent treatments of functional disorders will be delivered using either GK 4C or Perfexion.

Rotavirus infection among infants with diarrhea in Pakistan.

Rotavirus was examined in 818 diarrheal stool samples collected in Karachi, Pakistan, from 1990 to 1997. Rotavirus was detected in 112 samples (13.7%). The predominant serotypes were G1 and G4 and G3 was not detected. The predominant type changed between years. Rotavirus was found in all seasons and most infections were found in children aged less than 2 years.

Epidemiology of poliomyelitis in Karachi, Pakistan: prospective studies during 1990-93.

Between October 1989 and September 1993, 245 cases of poliomyelitis visited the Department of Pediatrics, Civil Hospital Karachi, Pakistan. The majority of them were between 6 months and 2 years of age and the epidemic occurred during the hot season. The dominant serotype was polio type 1. All of the polioviruses isolated from the patients were wild type. Virological studies also disclosed that enteroviruses other than polioviruses were prevalent among healthy children as well as diarrheal and polio patients. Serodiagnosis by poliovirus-specific immunoglobulin M antibody tests using the capture enzyme-linked immunosorbent assay method were in good agreement with the results of virus isolation. The present study demonstrated that Pakistan is a region endemic for wild poliovirus and more aggressive preventive measures are needed to eradicate poliomyelitis from the region.

Isolation of cytopathic small round virus (Aichi virus) from Pakistani children and Japanese travelers from Southeast Asia.

Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.

Virological and serological studies on poliomyelitis in Karachi, Pakistan. I. Outbreaks in 1990-91.

Between October 1989 and September 1991, 124 cases of poliomyelitis visited the Department of Paediatrics, Civil Hospital Karachi, Pakistan. The majority of them were between 6 months and 2 years of age and the epidemics occurred during the hot seasons. The dominant serotype was poliovirus type 1 during the epidemic season in 1990 and type 2 in 1991. All the polioviruses isolated from the patients were wild-type. Virological studies also disclosed that enteroviruses other than polioviruses were prevalent among healthy children as well as among diarrheal and polio patients. A serological survey to elucidate the serological efficacy of oral polio vaccine (OPV) showed that: (i) in 112 unimmunized children, after disappearance of transplacental maternal antibody during early infancy, antibody prevalence increased gradually and > 80% of the children were seropositive against all three types of polioviruses at 5 years of age; (ii) in 201 children immunized with full doses of OPV in their infancy, the decrease in antibody titer during infancy was less and seroprevalence rose sharply afterwards: at 2 years of age, > 80% of them were seropositive against all three types of the virus. The rapid increase of seroprevalence might be the effect of OPV administration. However, the prevalence was lower than that in developed countries.