Analysis of extracorporeal photopheresis within the frame of the WAA register.

The aim of the study was to investigate safety and if extracorporeal photopheresis (ECP) may change health criteria (HC) and quality of life (QoL). MATERIAL AND METHOD: 560 patients (33 % women) were treated with ECP for a total of 13,871 procedures during a 17-years period. Mean age was 48 years (±18, range 3-81 years). Self-estimation of QoL was graded: 0 (suicidal) up to 10 (best ever) and HC: 0 (Bed ridden, ICU condition) up to 10 (athletic). Adverse events were analyzed. ANOVA and paired comparisons were performed. RESULTS: Patients were treated due to graft versus host disease (GVHD, n = 317), skin lymphoma (n = 70), solid organ transplants (n = 47), skin diseases (n = 20) and other diseases (n = 106). Adverse events (AEs) were registered in 5.4 % of the first treatments and in 1.2 % of the subsequent procedures. Severe AEs were present in 0.04 % of all procedures. No patient died due to the procedure. Tingling and stitching were the most common AE. For those with GVHD an improvement was noticed within approximately 10 procedures of ECP in the severity stage, QoL (from a mean of 6.1 to 6.8, p < 0.002) and the HC (6.1 -> 6.4, p < 0.014) and improved further with added procedures. CONCLUSION: Photopheresis is an established therapy with few side effects. The present study of soft variables indicate that GVHD shows benefits upon ECP within approximately 10 procedures in regard to the severity of mainly skin GVHD, and lower baseline levels of HC and QoL.

Analyses of registry data of patients with anti-GBM and antineutrophil cytoplasmatic antibody-associated (ANCA) vasculitis treated with or without therapeutic apheresis.

Therapeutic apheresis (TA) as a treatment for antibody-associated vasculitis (AAV) was questioned by the PEXIVAS although the MEPEX study favored TA. The aim of this study was to evaluate the efficacy of TA to improve renal function in patients consecutively included in the WAA-apheresis registry versus patients not treated with TA. MATERIALS AND METHODS: Included were 192 patients that suffered from anti-glomerular basement membrane disease (anti-GBM, n = 28) and antineutrophil cytoplasmic antibody-associated vasculitis of MPO or PR3 origin. Of these 119 had performed TA and the other 73 had not performed TA for theses diagnoses (CTRL). RESULTS: Elderly had an increased risk to die within 12 months (p = 0.002). All 28 anti-GBM had renal involvement, 21 dialysis dependent. At 3 month nine (36 %) did not need dialysis. Baseline data regarding renal function of AAV patients, subtype MPO and PR3, were worse in the TA groups than in CTRL. Recovery out of dialysis was better for the PR3-TA group compared with 1) the controls of MEPEX (RR 0.59, CI 0.43-0.80) and 2) the MPO-TA patients (RR 0.28, CI 0.12-0.68). The MPO-TA recovered similarly as the MEPEX-CTRL. Renal function improved most for TA-patients from baseline during the first 3 months (MPO-TA and PR3-TA) and stabilized thereafter and less for MPO-CTRL and PR3-CTRL. CONCLUSION: PR3-TA patients seem to have best chances to get out of dialysis. PR3-TA and MPO-TA improved residual renal function better than CTRL. The present study recommends reconsiderations to use TA for AAV especially those with PR3-vasculitis with severe renal vasculitis.

Evaluation of health and quality of life in apheresis patients - data from the WAA register.

OBJECTIVE: The World Apheresis Association (WAA) register contains data from more than 89 000 apheresis procedures in more than 12,000 patients. The aim of this study was to evaluate functional health and quality of life (QoL) in patients during apheresis treatment. MATERIAL AND METHODS: Estimates of health condition (HC) were made in 40,445 and of QoL in 22112 apheresis procedures. This study focused on a 10-step graded evaluation of HC (scale from: 'bedridden, unable to eat' to a level of 'athletic competition') and self-assessment of QoL (scale from: worst ever '0' to best ever '10'). Data were compared in relation to various apheresis procedures and if the patient underwent the first or subsequent apheresis procedure. RESULTS: Of the patients treated with plasma exchange (PEX) with centrifugation technique (n = 15787) 10% were 'bedridden, unable to come out of bed' while for patients treated with plasma filtration technique (n = 1018) the percentage was 27%. During the first procedure these figures were 16% and 30%, respectively. Self-estimates of QoL were graded 'zero' or '1' in 1.6% of patients during the first apheresis procedure; At the first contact patients undergoing PEX graded like this in 4.3%. CONCLUSION: Many of the patients undergoing apheresis treatment have poor HC and QoL at the start of therapy. Of all therapeutic apheresis procedures patients undergoing PEX had the lowest score of QoL.

Distribution of indications and procedures within the framework of centers participating in the WAA apheresis registry.

The WAA apheresis registry was established in 2003 and an increasing number of centers have since then included their experience and data of their procedures. The registry now contains data of more than 74,000 apheresis procedures in more than 10,000 patients. This report shows that the indications for apheresis procedures are changing towards more oncological diagnoses and stem cell collections from patients and donors and less therapeutic apheresis procedures. In centers that continue to register, the total extent of apheresis procedures and patients treated have expanded during the latest years.

Relevance of flow cytometric enumeration of post-thaw leucocytes: influence of temperature during cell staining on viable cell recovery.

BACKGROUND AND OBJECTIVES: Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples, potentially due to different temperatures during cell staining. MATERIALS AND METHODS: Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients, and from 10 paediatric patients. After thawing, cells were prepared for analysis within 30 min between thawing and acquisition, at either 4°C or at room temperature. RESULTS: For cell products cryopreserved in conventional freezing medium (10% final DMSO), viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed, CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT), followed by CD3, CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO), median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS: The post-thaw environment and, potentially, the cryoprotectant impact the outcome of cell enumeration, and results from the analysis tube may not be representative of the cells infused into a patient.

Adverse events in apheresis: An update of the WAA registry data.

Apheresis with different procedures and devices are used for a variety of indications that may have different adverse events (AEs). The aim of this study was to clarify the extent and possible reasons of various side effects based on data from a multinational registry. The WAA-apheresis registry data focus on adverse events in a total of 50846 procedures in 7142 patients (42% women). AEs were graded as mild, moderate (need for medication), severe (interruption due to the AE) or death (due to AE). More AEs occurred during the first procedures versus subsequent (8.4 and 5.5%, respectively). AEs were mild in 2.4% (due to access 54%, device 7%, hypotension 15%, tingling 8%), moderate in 3% (tingling 58%, urticaria 15%, hypotension 10%, nausea 3%), and severe in 0.4% of procedures (syncope/hypotension 32%, urticaria 17%, chills/fever 8%, arrhythmia/asystole 4.5%, nausea/vomiting 4%). Hypotension was most common if albumin was used as the replacement fluid, and urticaria when plasma was used. Arrhythmia occurred to similar extents when using plasma or albumin as replacement. In 64% of procedures with bronchospasm, plasma was part of the replacement fluid used. Severe AEs are rare. Although most reactions are mild and moderate, several side effects may be critical for the patient. We present side effects in relation to the procedures and suggest that safety is increased by regular vital sign measurements, cardiac monitoring and by having emergency equipment nearby.

Analyses of data of patients with Thrombotic Microangiopathy in the WAA registry.

UNLABELLED: Thrombotic Microangiopathy (TMA) is a histopathological feature of various diseases including thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. The aim of this study was to investigate the outcome and prognostic variables of TMA-patients. MATERIALS AND METHODS: Data were consecutively retrieved from the WAA-apheresis registry (www.waa-registry.org) during 2003-2009. Included were all 120 patients (1237 procedures) who suffered from various forms of TMA, as registered by the ICD-10 code M31.1. Besides registry data, more extensive information was retrieved from the latest 64 patients. Adverse events of the TMA patients were compared to those of the other patients in the registry. RESULTS: The mean age was 46 years (range 11-85 years, 57% women). In 72% therapeutic apheresis was due to an acute indication while a long-term indication was present in 28%. Plasma exchange was performed by centrifugation and filtration technique (95% and 4%, respectively), and immunoadsorption in 1% of the patients. Only fresh frozen plasma was used as replacement fluid in 69% of procedures. Adverse events were more frequent than in the general apheresis population (10% versus 5%, RR 1.9, CI 1.6-2.3). No death occurred due to apheresis treatment. Three percent of the procedures were interrupted. Bronchospasm and/or anaphylactic shock were present in two patients and one patient suffered from TRALI. At admission 26% were bedridden and needed to be fed. The risk of dying during the treatment period was significantly higher if the patient also suffered from a compromising disease, such as cancer. There was an inverse correlation between the ADAMTS13 level and the antibody titer (r=-0.47, p=0.034). CONCLUSIONS: Patients with TMA have an increased risk for moderate and severe AE compared to the general apheresis population. Many patients were severely ill at admission. The prognosis is worse if the patient also has a severe chronic disease. Even slightly increased ADAMTS13-antibody titers seem to have a negative impact on the ADAMTS13 levels.

Thrombotic microangiopathy.

Thrombotic microangiopathy (TMA) is a histopathological feature of various diseases including thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). There are many secondary causes of TMA, many of them could mimic TTP or HUS. This article presents a short overview on TMA. In conclusion TMA is the result of various etiology reasons and pathologic reactions with various clinical entities. It is important to focus on a thorough history including family history when deciding on a diagnosis. Analysis of ADAMTS 13 and ADAMTS 13-antibodies may help to decide continued therapy.

Bone marrow processing with the AMICUS™ separator system.

UNLABELLED: ABO incompatible bone marrow transplantation (BMT) requires processing of the donated bone marrow (BM), either erythrocyte depletion, or also a volume reduction. The AMICUS™ system was introduced in the field of peripheral blood mononuclear cell collection, showing a good performance regarding efficiency and safety. To evaluate the performance of the MNC collection program of the Amicus device for BM, we analysed our data obtained from the Amicus and the Fenwal CS3000omnix™ plus device. METHODS: From 2005 to 2008, we performed 22 automated erythrocyte depletions of BM for ABO mismatched BMT in 21 patients, 11 with the Amicus (A; 10 patients) and 11 with the CS3000 (F; 11) device. RESULTS: There were no statistical differences in donor age, recipient age, type of ABO mismatch, and CD34+ cell yield [group A pre 7.03 post 4.93 vs. group F pre 8.55 and post 6.2 × 10E06 cells per kilogram of bodyweight] for both devices. The efficiency for the CD34+ cell collection was lower, but not statistically significant, in the Amicus device (70% ± 12 vs. 84% ± 12; U-test P = 0.123). The erythrocyte volume in the final product was higher but not statistically significant different in the Amicus device (9.46 ± 2.3 vs. 6.98 ± 3.3 ml; U-test P = 0.17). During the evaluation period, no technical problems were observed. All patients but one, who died at d + 11, showed a sustained engraftment. CONCLUSIONS: We conclude that, in principle, the Amicus device can be used for MNC collection from BM to deplete erythrocytes from BM grafts in allogeneic stem cell transplantations.

Compassionate use of bevacizumab (Avastin) in children and young adults with refractory or recurrent solid tumors.

BACKGROUND: The aim of this study was to evaluate feasibility and toxicity of bevacizumab (Avastin), a monoclonal antibody directed against the vascular endothelial growth factor in children and young adults. PATIENTS AND METHODS: Fifteen patients (male: n = 8; female: n = 7; median age, 14.6 years) received bevacizumab for recurrent or progressive solid tumors (carcinoma: n = 3; neuroblastoma: n = 2; astrocytoma grade III: n = 2; rhabdomyosarcoma: n = 2; nephroblastoma: n = 2; benign vascular tumors: n = 2; synovial sarcoma: n = 1; and malignant hemangiopericytoma: n = 1) on a compassionate basis. Bevacizumab was administered at 5-10 mg/kg body weight intravenously every 2-3 weeks. Most patients received chemotherapy in addition to bevacizumab. Duration of bevacizumab therapy ranged from 1.5 to 23 months. RESULTS: Bevacizumab-related side-effects were mild and included hypertonia (n = 2), proteinuria/hematuria (n = 2), epistaxis (n = 2), local erythema (n = 1), and defective wound healing and ascites (n = 1). Radiographic objective responses (partial responses) were observed in two patients with astrocytoma grade III and in one patient each with neuroblastoma and pleomorphic rhabdomyosarcoma, respectively. CONCLUSIONS: Bevacizumab seems to have a good acute safety profile and some antitumor activity in heavily pretreated children and young adults with recurrent solid tumors. Prospective clinical trials are urgently needed to further evaluate the safety and efficacy of bevacizumab in pediatric patients.

Detection of human cytomegalovirus-specific T lymphocytes in human blood: comparison of two methods.

BACKGROUND: Human cytomegalovirus (HCMV) infection remains a major cause of morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation (SCT). In the case of HCMV reactivation, the well-defined detection of virus-specific effector cells in patients might positively impact antiviral treatment. METHODS: We examined blood samples from healthy volunteers serologically typed for HCMV IgG. Based on multicolor flow cytometry analysis, we addressed HCMV-specific CD8(+) effector T lymphocytes using HCMV-specific tetramers for the respective major histocompatibility complex (MHC) class I type. As a second approach, we employed the cytokine secretion assay (CSA), which allows the indirect detection of target-specific CD4(+) and CD8(+) T cells via their interferon (IFN)-gamma secretion upon HCMV pp65 in vitro stimulation. RESULTS: We hypothesized the detection of HCMV-specific lymphocytes in >50% of healthy Caucasians that were IgG-seropositive for HCMV. In terms of specificity, both assays showed comparably good results (specificity 100%, confidence interval >95%). Regarding sensitivity, both assays met the zero hypothesis. However, with 45/52 (86.5%) the tetramer technology was superior to the CSA, which detected 34/52 (65.4%) based on CD8(+) T cells and 41/52 (78.8%) based on both CD4(+) and CD8(+) T cells. DISCUSSION: A good correlation was observed between both assays, although the tetramers addressed only CD8(+) HCMV-specific T cells, whereas IFN-gamma secretion was detected on all T-cell types. Disadvantages of the CSA are the time-consuming stimulation, the extensive cell washing steps and the fact that the target cells are detected indirectly. The analysis with tetramers is rapid and reliable but their general use is hampered because of the restriction to a few HLA types.

World apheresis registry 2003-2007 data.

OBJECTIVES: Seventy-five centers from many countries have applied for a login code to the WAA apheresis registry. Fifteen centers from 7 countries have been actively entering data at the internet site from 2003 until 2007. We report on data from the registry so far. METHODS: This is a web-based registry. A link is available from the WAA homepage (www.worldapheresis.org). So far data from 2013 patients (12,448 procedures) have been included. A median of 6 treatments have been performed (range 1-140). Mean age 51 years (range 1-94 years; 45% women). Seven percent of the patients were < or = 21 years and 4% were < or = 16 years. RESULTS: The purpose of the apheresis procedure was therapeutic in 67% and retrieval of blood components in 33%. Main indications: neurological and hematological diseases, lipid apheresis and stemcell collection (autologous, and some allogeneic). Blood access: peripheral vessels (71%), central dialysis catheter through jugular (6.5%) or subclavian veins (6.7%), femoral vein (8%) and AV fistula (4%). ACD was used for anticoagulation in 73% of the procedures. Albumin was mainly used as replacement fluid. Adverse events (AE) were registered in 5.7% of the procedures. AE was graded as mild (2.5%), moderate (2.7%) or severe (0.5%). No death occurred due to treatment. The procedures were interrupted in 2.6%. Most frequent AEs were blood access problems (29%), tingling around the mouth (20%), hypotension (18%), and urticaria (9%). There were significant differences between the centers regarding mild and moderate AEs. Data indicate that centers using continuous infusion of calcium had fewer AEs. CONCLUSION: There was a limited number of severe AEs. Centers use various standard procedures for apheresis. By learning from the experience of others the treatment quality will improve further. In the near future, an update of the registry will enable more extensive evaluation of the data.

Phase I study of tumor Ag-loaded IL-12 secreting semi-mature DC for the treatment of pediatric cancer.

BACKGROUND: Cancer vaccines employing DC in their capacity as APC have been tolerated well and have shown some efficacy in clinical studies. IL-12, a cytokine critical for type 1 T-helper (Th1) lymphocyte and cytotoxic T-lymphocyte (CTL) differentiation, when released from a DC-based cancer vaccine, may support the generation of a cellular T-cell response. METHODS: We applied tumor cell lysate plus keyhole limpet hemocyanin (KLH)-loaded and 48-h lipopolysaccharide (LPS) plus IFN-gamma-stimulated fully mature DC, which do not release IL-12, subcutaneously to eight patients, and maximally 6-h stimulated semi-mature (sm) DC, which are potent producers of IL-12, subcutaneously (n=6) or intranodally (n=8) as a cancer vaccine to patients suffering from advanced solid pediatric malignancies. RESULTS: No serious adverse events were observed following application of IL-12-releasing smDC. Following immunization the majority of patients responded positively to KLH in a delayed-type hypersensitivity (DTH) test. In addition, three of six intranodally treated patients responded to the tumor Ag in the DTH test. DISCUSSION: We conclude that treatment with a DC-based cancer vaccine enabled to release the immune regulatory cytokine IL-12 is safe and feasible and has the potential to induce a cellular immune response in pediatric cancer patients.

Volcanic ash supports a diverse bacterial community in a marine mesocosm.

Shallow-water coral reef ecosystems, particularly those already impaired by anthropogenic pressures, may be highly sensitive to disturbances from natural catastrophic events, such as volcanic eruptions. Explosive volcanic eruptions expel large quantities of silicate ash particles into the atmosphere, which can disperse across millions of square kilometres and deposit into coral reef ecosystems. Following heavy ash deposition, mass mortality of reef biota is expected, but little is known about the recovery of post-burial reef ecosystems. Reef regeneration depends partly upon the capacity of the ash deposit to be colonised by waterborne bacterial communities and may be influenced to an unknown extent by the physiochemical properties of the ash substrate itself. To determine the potential for volcanic ash to support pioneer bacterial colonisation, we exposed five well-characterised volcanic and coral reef substrates to a marine aquarium under low light conditions for 3 months: volcanic ash, synthetic volcanic glass, carbonate reef sand, calcite sand and quartz sand. Multivariate statistical analysis of Automated Ribosomal Intergenic Spacer Analysis (ARISA) fingerprinting data demonstrates clear segregation of volcanic substrates from the quartz and coral reef substrates over 3 months of bacterial colonisation. Overall bacterial diversity showed shared and substrate-specific bacterial communities; however, the volcanic ash substrate supported the most diverse bacterial community. These data suggest a significant influence of substrate properties (composition, granulometry and colour) on bacterial settlement. Our findings provide first insights into physicochemical controls on pioneer bacterial colonisation of volcanic ash and highlight the potential for volcanic ash deposits to support bacterial diversity in the aftermath of reef burial, on timescales that could permit cascading effects on larval settlement.

Transplantation of highly purified peripheral blood CD34+ cells from HLA-mismatched parental donors in 14 children: evaluation of early monitoring of engraftment.

HLA-mismatched family members may represent an important cell source for patients that require stem cell transplantation but lack both a matched sibling donor and a closely matched unrelated donor. We report the outcome of 19 transplantations from HLA two- or three- loci mismatched parental donors in which 14 pediatric patients with hematological malignancies or other disorders, received a median of 21.5 x 106 (range, 5.4-58) highly purified CD34+peripheral blood stem cells (PBSC), as well as 4.7 x 104 (range, 0.4-12) donor T cells per kg body weight. T cell depletion was performed using a two-step CD34-positive selection on two different magnetic beads devices. Ten of 14 patients presented with rapid myeloid engraftment. The four patients who presented with graft failure (two non-engraftments, two rejections) received a second stem cell graft and one a third. Graft rejection was detected early by polymerase chain reaction (PCR) analysis of FACS-sorted T cells. Eight of the 14 patients are still alive after a median observation period of 15. 6 months (range, 3-31.3) with full donor chimerism in all hematopoietic cell lineages. No acute organ graft-versus-host disease (GVHD) and no chronic GVHD have occurred. One patient experienced relapse of leukemia. We conclude that transplantation of allogeneic PBSC from haploidentical donors will open new perspectives for pediatric patients for whom an HLA-matched stem cell graft is not available. Close monitoring of recipient and donor hematopoiesis might be of clinical value, to recognize early engraftment or rejection.

Resolution of early cytomegalovirus (CMV) infection after leukocyte transfusion therapy from a CMV seropositive donor.

A 2 year and 8 month old CMV-negative boy suffering from stage III neuroblastoma underwent ABMT in first very good partial remission. He acquired early CMV infection on day +5, followed by consecutive graft failure and severe sepsis, and the clinical course deteriorated. Between days +16 and +21, he received seven leukocyte concentrates (LC) collected from a healthy, but CMV-IgG-seropositive relative stimulated with G-CSF (filgastrim, 5 microg/kg/day). A median of 5.7 x 10(10) neutrophils/m2/day (range, 1.2-8.3) were transfused, corresponding to a T cell number of roughly 4 x 10(8) CD3+ cells/kg/day. After infusion of the LCs, PCR analysis became negative for CMV and the patient received his rescue bone marrow. One year after ABMT, he is in complete remission and in good clinical condition. Our results suggest that the T cells infused together with the irradiated leukocytes played a major role in eradicating the CMV infection in this patient.

Low incidence of molecular evidence for tumour in PBPC harvests from patients with high risk Ewing tumours.

Reverse transcriptase polymerase chain reaction (RT-PCR) was applied to evaluate the frequency of tumour cells in PBPC products from 15 high risk Ewing tumour (ET) patients who were treated according to EICESS 92 with high-dose chemotherapy (HDC) and stem cell rescue. Initial tumour cell contamination of the bone marrow (BM) detected by light microscopy was found in five and by RT-PCR in eight cases. RT-PCR was performed on each PBPC sample repeatedly at a sensitivity comparable to 20-100 highly EWS-Fli1 expressing tumour cells per 10 ml of fresh blood. Irrespective of the extent of BM involvement at diagnosis, all BM samples obtained before harvest were RT-PCR negative. Among 12 of 35 analysed apheresis products with single positive RT-PCR results only one sample tested reproducibly positive for tumour cell contamination in independent determinations. These preliminary data suggest that tumour cell contamination of PBPC is rarely found in patients with ET.

Permanent tunneled silicone central venous catheters for autologous PBPC harvest in children and young adults.

Children with high risk malignancies are usually given permanent (Hickman-type) tunneled silicone rubber central venous catheters (silicone CVCs) for the administration of chemotherapy. In the past, these children received an additional short-term polyurethane dialysis CVC for stem cell apheresis. To avoid placement of an additional short-term CVC, we started in 1995 to use pre-existing silicone CVCs for PBPC harvests. From May 1996 to February 1999 we evaluated 165 harvests in 37 children and 14 young adults (16-28 years) treated with high-dose chemotherapy and stem cell support, comparing CD34+ cell harvest efficiency, catheter tolerability, and complications in three different approaches to vascular access. Pre-existing silicone CVCs (64%) or peripheral venous cannulae (15%) were the first choice for venous access. Only when these failed were polyurethane CVCs (21%) used. No significant difference was seen between these three groups, even after dividing the silicone CVC group (105 harvests in 32 patients) into three subgroups according to weight and age. The most frequent problems were citrate toxicity (n = 33), mechanical obstruction inside (n = 9) and outside the cell separator (n = 2), decreased draw line flow in silicone CVCs (n = 7), decreased draw line flow in peripheral venous cannulae (n = 6), and one occlusion in a polyurethane CVC. Pre-existing CVCs and peripheral venous cannulae functioned efficiently when used as a draw line in 79% of the apheresis procedures without significantly reducing single harvest efficiency or catheter tolerability. Consequently, the risks and costs associated with the placement of a dialysis CVC could be avoided in the majority of cases. Bone Marrow Transplantation (2000) 26, 781-786.

Environmental health conditions and cholera vulnerability in Latin America and the Caribbean.

PIP: Epidemic cholera reached South America in January 1991 and later spread to Central America and the United States. It afflicted 312,000 people and claimed 3200 lives. Since cholera had not been in Latin America for almost 70 years, health authorities allowed environmental health barriers to cholera collapse. For example, the Governments of the Region agreed in 1961 to abide by the Charter of Punta del Este to provide water and sewerage to 70% of the urban population and 50% f the rural population by 1971. They did not achieve their goals for the rural population. In fact, at the end of 1988, water was piped to 79% of the urban households and an additional 11% of the urban population had access to a public water source. Sewerage services served 49% of the urban population and, with other methods of excreta disposal, 80% of the population had adequate excreta disposal. On the other hand, only 55% of rural inhabitants had access to either piped water or public standpipes. Further sanitary excreta disposal services only covered 32%. Besides the water quality of existing water supply systems was poor. Since feces of infected people have as many as 1 billion Vibrio cholerae and , in some of Vibrio, up to 80% of carriers exhibit only mild symptoms or no symptoms at all, it is easy to understand how cholera took hold in Latin America. Researchers identified the points of contamination responsible for the cholera outbreak in Piura and Trujillo, Peru to be wells, distribution systems, and house. Annual population growth in Latin America at 2.6% poses specific problems to providing enough water and sanitation services to all in need, especially those in marginal areas around the cities (who will make up 40% of the population by 2000).^ieng