Alexandre Macedo de Oliveira

An Outbreak of Hepatitis C Virus Infections among Outpatients at a Hematology/Oncology Clinic

Context Hepatitis C virus (HCV) may be transmitted through health careassociated exposure involving poor aseptic technique. Contribution In an outpatient hematology/oncology clinic, 99 patients who did not have previously known HCV infection acquired the virus, apparently because a health care worker reused contaminated syringes and saline bags. Cautions Researchers may have missed some cases because the investigation occurred more than a year after the outbreak. Implications We need active, effective infection-control programs for outpatient care settings. The Editors Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease in the United States (1). It is transmitted primarily through percutaneous exposure to contaminated blood (2). Health careassociated HCV transmission has been attributed to breaches in aseptic technique (3-5). In September 2002, a gastroenterologist notified the Office of Epidemiology at the Nebraska Health and Human Services System in Lincoln, Nebraska, of a cluster of 4 HCV genotype 3a infections in patients who received care at a single outpatient clinic in eastern Nebraska. Because these patients reported no typical risk factors for HCV infection and genotype 3a accounts for less than 8% of HCV infections in the United States, we suspected health careassociated transmission (1). The implicated facility was an independently owned and operated hematology/oncology clinic located inside a hospital complex. The clinic opened in January 1998, and approximately 500 patient visits occurred per month. The staff comprised an oncologist, a registered nurse, a certified nurse assistant, and a secretary. In October 2002, 1 month after the gastroenterologists report, the clinic voluntarily closed. We compared clinic patient lists with Nebraskas HCV, HIV, and hepatitis B virus (HBV) registries and identified a patient with preexisting chronic HCV genotype 3a infection who enrolled at the clinic in March 2000. We found no evidence of HIV or HBV transmission. Preliminary interviews with clinic staff revealed that the nurse who had worked at the clinic since its opening was dismissed in July 2001 because of breaches in infection- control practices. We initiated an investigation to confirm the hypothesis of health careassociated transmission and to determine the extent of the outbreak and its mechanism of transmission. Methods Case Finding and Laboratory Testing Using clinic records, we identified all patients who visited the clinic from March 2000 through December 2001. We contacted all living patients and offered them free HCV testing. Structured in-person interviews were conducted in a private setting with participating patients to assess their medical history, including previous hepatitis diagnosis and risk factors for HCV infection. All specimens were tested for HCV antibodies by using enzyme immunoassay (EIA) (Abbott HCV EIA 2.0, Abbott Laboratories, Abbott Park, Illinois). Positive results on EIA were confirmed by using recombinant immunoblot assay (RIBA) (Chiron RIBA HCV 3.0 SIA, Chiron Corp., Emeryville, California) or HCV qualitative polymerase chain reaction (PCR) (Cobas Amplicor HCV Test v2.0, Roche Molecular Diagnostic Systems, Branchburg, New Jersey) (6). Because of concerns that immunosuppressed patients could have impaired antibody response, we also tested specimens from patients seen before July 2001 by using a transcription-mediated amplification (TMA) assay (Gen-Probe Inc., San Diego, California). A positive HCV test result was defined as a positive finding on TMA or a confirmed positive finding on EIA. Samples that were positive on PCR or TMA were genotyped by using gene sequencing of the 5 untranslated region (PE Applied Biosystems, Foster City, California). Epidemiologic Investigation We reviewed and abstracted the medical records and medication sheets of the tested patients using structured forms to identify clinic-associated risk factors for HCV transmission, such as number of clinic visits, presence of a central venous catheter, and percutaneous exposures. A case was defined as a positive HCV test result in a clinic patient treated from March 2000 through December 2001 who did not have evidence of preexisting HCV infection (that is, abnormal alanine aminotransferase [ALT] levels or positive HCV test results) before enrollment at the clinic. We considered the date of HCV infection onset to be the date when an ALT level greater than 3 times the upper limit of normal was first recorded (7). We conducted a cohort analysis to evaluate the association between exposures at the clinic and HCV infection. To further evaluate the variables associated with HCV infection in the cohort analysis and to investigate potential confounding among these variables, we performed an analysis that focused on risk factor exposure during the period of probable transmission (Appendix). Review of Infection-Control Practices The physician and nurse who worked at the clinic during the outbreak period were unavailable or unwilling to submit to interviews and HCV testing. We interviewed other health care workers and patients to corroborate staff adherence to infection-control standards. Statistical Analysis We made comparisons by using the t-test, the Fisher exact test, or the chi-square test, as appropriate (Epi Info, version 3.01, 2003, Centers for Disease Control and Prevention, Atlanta, Georgia). P values less than 0.05 were considered statistically significant. Results Case Finding and Laboratory Testing A total of 842 patients attended the clinic from March 2000 through December 2001. We contacted the 613 (73%) living patients; 494 of these (81%) agreed to testing. Twenty-one tested patients whose medical charts could not be located were excluded from analysis. No cases were seen among the 103 tested patients who began treatment after the nurses dismissal in July 2001. Therefore, we defined our study period as March 2000 to July 2001 and considered only exposures that occurred during this period. Among the 370 eligible patients, 101 tested positive for HCV: 80 were EIA positive and PCR positive; 18 were EIA negative but TMA positive; and 3 were EIA positive, PCR negative, and RIBA positive. One patient with inconclusive HCV test results (positive EIA, negative PCR and TMA, and indeterminate RIBA results) was excluded from analysis. We also excluded 2 persons with evidence of preexisting HCV infection. One visited the clinic only once, in March 2001, and had elevated ALT levels before that visit. The other person was the presumed outbreak source-patient identified during our preliminary investigation. This patient enrolled at the clinic in March 2000 with preexisting chronic HCV genotype 3a infection (viral load > 200000 copies/mL); a central venous catheter was implanted at that time. The resulting cohort of 367 patients seen at the clinic from March 2000 to July 2001 consisted of 99 HCV-positive patients who lacked evidence of preexisting HCV infection and met the case definition and 268 HCV-negative patients. The overall attack rate was 27% (99 of 367 patients). Genotype 3a was identified in 95 (96%) cases; in 4 cases, low levels or absence of HCV RNA precluded genotype determination. Epidemiologic Investigation Descriptive Epidemiology Most infected patients were female (60%), and the median age was 66 years (range, 21 to 95 years). Ninety-five of the 99 patients (96%) had cancer as an underlying disease, and the median number of clinic visits was 21 (range, 3 to 78 visits). Signs and symptoms of acute HCV infection were uncommon: Four (4%) patients had clinical jaundice, and 16 (16%) reported nausea. We estimated an onset date of infection for 56 (57%) case-patients (Figure). Only 2 (2%) case-patients exhibited spontaneous viral clearance (that is, undetectable HCV RNA) at the time of the investigation. Figure. Number of cases of hepatitis C virus infection, by estimated onset date ( n = 56) (Nebraska, March 2000July 2001). Eighty-three (84%) infected patients had a central venous catheter in place while receiving care at the clinic. Twenty-six (26%) infected patients did not receive any intravenous drug at the clinic during the outbreak period, but did have catheters flushed with saline solution. All 99 patients who developed HCV infection visited the clinic the same day as a previously HCV-infected patient and had saline flushes on those days. Analytic Epidemiology Among the 367 patients in the cohort analysis, 140 (38%) received 1 or more saline flushes during the study period (Table 1). Of those, 99 (71%) became infected, compared with no infection among the 227 patients who did not receive a saline flush during that time (relative risk, undefined; P< 0.001). Bivariate analysis also indicated an association between HCV infection and 5 other variables: sex, underlying diagnosis, presence of central venous catheter, exposure to subcutaneous injections, and number of clinic visits. Table 1. Attack Rates of Hepatitis C Virus Infection, by Demographic and Selected Clinical Characteristics, among 367 Tested Patients at the Hematology/Oncology Clinic, March 2000July 2001 In the multivariable analysis using the case-patients with estimated date of onset, only the number of saline flushes remained significantly associated with HCV infection in our model (adjusted odds ratio, 2.1 [95% CI, 1.3 to 3.2]) (Table 2; methods described in Appendix). Of note, the 10 case-patients with the earliest onset dates all received a saline flush on a day when the source-patient visited the clinic (Appendix Figure). Appendix Figure. Timeline of clinic visits for the 10 case-patients with the earliest dates of onset and for the source-patient (Nebraska, March 2000July 2001). Table 2. Unconditional Multivariable Analysis of Risk Factors for Hepatitis C Virus Infection Using the 56 Case-Patients with Estimated Dates of Onset and 56 Controls, March 2000July 2001 Review of Infection-Control Practices Our i

Transfusion‐associated transmission of West Nile virus, United States 2003 through 2005

BACKGROUND: National blood donation screening for West Nile virus (WNV) started in June 2003, after the documentation of WNV transfusion‐associated transmission (TAT) in 2002.

West Nile Virus Blood Transfusion-Related Infection Despite Nucleic Acid Testing

BACKGROUND:  A case of West Nile virus (WNV) encephalitis associated with transfusion of blood that did not react when tested for WNV by minipool (MP) nucleic acid testing (NAT) is described. A Nebraska man developed clinical encephalitis 13 days after surgery and transfusion of 26 blood components. Antibody testing confirmed WNV infection. An investigation was initiated to determine the source of this infection.

South American Plasmodium falciparum after the Malaria Eradication Era: Clonal Population Expansion and Survival of the Fittest Hybrids

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradications successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999–2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006–2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.

Decline in Sulfadoxine-Pyrimethamine-Resistant Alleles after Change in Drug Policy in the Amazon Region of Peru

ABSTRACT The frequency of alleles with triple mutations conferring sulfadoxine-pyrimethamine (SP) resistance in the Peruvian Amazon Basin has declined (16.9% for dhfr and 0% for dhps compared to 47% for both alleles in 1997) 5 years after SP was replaced as the first-line treatment for Plasmodium falciparum malaria. Microsatellite analysis showed that the dhfr and dhps alleles are of common origin.

Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times.

Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites.

A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative isolates in the Colombian Amazon may have implications for the use of PfHRP2-based RDTs in the region and may explain inconsistencies observed when PfHRP2-based tests and assays are performed.

Ownership and usage of insecticide-treated bed nets after free distribution via a voucher system in two provinces of Mozambique

BackgroundInsecticide-treated bed nets (ITNs) are an efficacious intervention for malaria prevention. During a national immunization campaign in Mozambique, vouchers, which were to be redeemed at a later date for free ITNs, were distributed in Manica and Sofala provinces. A survey to evaluate ITN ownership and usage post-campaign was conducted.MethodsFour districts in each province and four enumeration areas (EAs) in each district were selected using probability proportional to size. Within each EA, 32 households (HHs) were selected using a simple random sample. Interviews to assess ownership and usage were conducted in each of the selected HHs using personal digital assistants.ResultsValid interviews were completed for 947 (92.5%) (440 in Manica and 507 in Sofala) of the 1,024 selected HHs. Among participating HHs, 65.0% in Manica and 63.1% in Sofala reported that at least one child under five years of age slept in the house the previous night. HH ownership of at least one bed net of any kind was 20.6% (95% confidence interval [CI]: 7.9%-43.6%) and 35.6% (95% CI: 27.8%-44.3%) pre-campaign; and 55.1% (95% CI: 43.6%-66.1%) and 59.6 (95% CI: 42.4%-74.7%) post-campaign in Manica and Sofala, respectively. Post-campaign HH ownership of at least one ITN was 50.2% (95% CI: 41.8%-58.5%) for both provinces combined. In addition, 60.3% (95% CI: 50.6%-69.2%) of children under five years of age slept under an ITN the previous night.ConclusionsThis ITN distribution increased bed net ownership and usage rates. Integration of ITN distribution with immunization campaigns presents an opportunity for reaching malaria control targets and should continue to be considered.

Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras.

BackgroundRecent studies have demonstrated the deletion of the histidine-rich protein 2 (PfHRP2) gene (pfhrp2) in field isolates of Plasmodium falciparum, which could result in false negative test results when PfHRP2-based rapid diagnostic tests (RDTs) are used for malaria diagnosis. Although primary diagnosis of malaria in Honduras is determined based on microscopy, RDTs may be useful in remote areas. In this study, it was investigated whether there are deletions of the pfhrp2, pfhrp3 and their respective flanking genes in 68 P. falciparum parasite isolates collected from the city of Puerto Lempira, Honduras. In addition, further investigation considered the possible correlation between parasite population structure and the distribution of these gene deletions by genotyping seven neutral microsatellites.MethodsSixty-eight samples used in this study, which were obtained from a previous chloroquine efficacy study, were utilized in the analysis. All samples were genotyped for pfhrp2, pfhrp3 and flanking genes by PCR. The samples were then genotyped for seven neutral microsatellites in order to determine the parasite population structure in Puerto Lempira at the time of sample collection.ResultsIt was found that all samples were positive for pfhrp2 and its flanking genes on chromosome 8. However, only 50% of the samples were positive for pfhrp3 and its neighboring genes while the rest were either pfhrp3-negative only or had deleted a combination of pfhrp3 and its neighbouring genes on chromosome 13. Population structure analysis predicted that there are at least two distinct parasite population clusters in this sample population. It was also determined that a greater proportion of parasites with pfhrp3-(and flanking gene) deletions belonged to one cluster compared to the other.ConclusionThe findings indicate that the P. falciparum parasite population in the municipality of Puerto Lempira maintains the pfhrp2 gene and that PfHRP2-based RDTs could be considered for use in this region; however continued monitoring of parasite population will be useful to detect any parasites with deletions of pfhrp2.

Comparison of molecular tests for the diagnosis of malaria in Honduras

BackgroundHonduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras.MethodsA total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species.ResultsOf the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite.ConclusionsMolecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.