Kimberly Baltzell

The Usefulness of Rapid Diagnostic Tests in the New Context of Low Malaria Transmission in Zanzibar

Background We assessed if histidine-rich-protein-2 (HRP2) based rapid diagnostic test (RDT) remains an efficient tool for Plasmodium falciparum case detection among fever patients in Zanzibar and if primary health care workers continue to adhere to RDT results in the new epidemiological context of low malaria transmission. Further, we evaluated the performance of RDT within the newly adopted integrated management of childhood illness (IMCI) algorithm in Zanzibar. Methods and Findings We enrolled 3890 patients aged ≥2 months with uncomplicated febrile illness in this health facility based observational study conducted in 12 primary health care facilities in Zanzibar, between May-July 2010. One patient had an inconclusive RDT result. Overall 121/3889 (3.1%) patients were RDT positive. The highest RDT positivity rate, 32/528 (6.1%), was found in children aged 5–14 years. RDT sensitivity and specificity against PCR was 76.5% (95% CI 69.0–83.9%) and 99.9% (95% CI 99.7–100%), and against blood smear microscopy 78.6% (95% CI 70.8–85.1%) and 99.7% (95% CI 99.6–99.9%), respectively. All RDT positive, but only 3/3768 RDT negative patients received anti-malarial treatment. Adherence to RDT results was thus 3887/3889 (99.9%). RDT performed well in the IMCI algorithm with equally high adherence among children <5 years as compared with other age groups. Conclusions The sensitivity of HRP-2 based RDT in the hands of health care workers compared with both PCR and microscopy for P. falciparum case detection was relatively low, whereas adherence to test results with anti-malarial treatment was excellent. Moreover, the results provide evidence that RDT can be reliably integrated in IMCI as a tool for improved childhood fever management. However, the relatively low RDT sensitivity highlights the need for improved quality control of RDT use in primary health care facilities, but also for more sensitive point-of-care malaria diagnostic tools in the new epidemiological context of low malaria transmission in Zanzibar. Trial registration ClinicalTrials.gov NCT01002066

Limited evidence of human papillomavirus on breast tissue using molecular in situ methods

Human papillomavirus (HPV) has been proposed as an etiologic agent of breast cancer based on numerous reports of high‐risk (oncogenic) HPV types in malignant breast tissues. However, most of those studies used standard and nested solution polymerase chain reaction (PCR) techniques, both of which are disadvantaged by vulnerability to laboratory contamination from positive control DNA and the inability to localize the signal to a specific cell type. To overcome these drawbacks, the authors of this report explored the use of in situ molecular methods of viral detection to reassess the frequency of HPV in malignant breast tissue.

A qualitative study to assess community barriers to malaria mass drug administration trials in the Gambia

BackgroundMass drug administration (MDA) is a strategy widely used in the control of human parasitic diseases but has been rarely attempted with malaria, the most common and dangerous parasitic disease in humans. MDA is an intervention strategy that involves simultaneously dispensing treatment to an entire population in a given geographic area. With some areas in sub-Saharan Africa documenting a decline in malaria transmission, the feasibility of MDA to further reduce malaria transmission is being considered. Understanding community perceptions of such an activity is vitally important for the design of the study and gaining the support of participants in order to maximize compliance and adherence.MethodsA qualitative study to assess factors likely to influence community acceptance of MDA in the seasonal and low malaria transmission setting of The Gambia was conducted. Using in-depth interviews, the perceptions, knowledge and attitudes of medical personnel and community members who have undergone MDA trials in The Gambia were investigated.ResultsSeveral major themes emerged, namely: 1) the importance of timing of rounds of MDA doses for maximum participation; 2) the need to educate the target population with accurate information on the procedures, drug regimen, and possible side effects to enhance adherence; 3) the need for continuous sensitization meetings to maintain and increase uptake of MDA; and, 4) the importance for defining roles in the delivery and assessment of MDA, including existing healthcare structures.DiscussionTo increase the likelihood of participation in MDA trials in this setting, activities should be undertaken just before and during the rainy season when community members are less mobile. Importantly, fears regarding blood sampling and side effects of the drug regimen need to be addressed prior to the start of the trial and repeated throughout the study period. Accurate and frequent communication is essential, and village leaders should consistently be included in sensitization meetings to enhance community participation. Additionally, village healthcare workers should be included in training and implementation, with supervision by a fieldworker permanently posted in every few villages during the trial. Future collaboration with Senegal may prove important for enhanced elimination efforts in The Gambia.

Febrile illness management in children under five years of age: a qualitative pilot study on primary health care workers¿ practices in Zanzibar

BackgroundIn Zanzibar, malaria prevalence dropped substantially in the last decade and presently most febrile patients seen in primary health care facilities (PHCF) test negative for malaria. The availability of rapid diagnostic tests (RDTs) allows rural health workers to reliably rule out malaria in fever patients. However, additional diagnostic tools to identify alternative fever causes are scarce, often leaving RDT-negative patients without a clear diagnosis and management plan. This pilot study aimed to explore health workers’ practices with febrile children and identify factors influencing their diagnostic and management decisions in non-malarial fever patients.MethodsSemi-structured key informant interviews were conducted with 12 health workers in six PHCFs in North A district, Zanzibar, April to June 2011. Interviews were coded using Atlas.ti to identify emerging themes that play a role in the diagnosis and management of febrile children.ResultsThe following themes were identified: 1) health workers use caregivers’ history of illness and RDT results for initial diagnostic and management decisions, but suggest caregivers need more education to prevent late presentation and poor health outcomes; 2) there is uncertainty regarding viral versus bacterial illness and health workers feel additional point-of-care diagnostic tests would help with differential diagnoses; 3) stock-outs of medications and limited caregivers’ resources are barriers to delivering good care; 4) training, short courses and participation in research as well as; 5) weather also influences diagnostic decision-making.ConclusionsThis pilot study found that health workers in Zanzibar use caregiver history of fever and results of malaria RDTs to guide management of febrile children. However, since most febrile children test negative for malaria, health workers believe additional training and point-of-care tests would improve their ability to diagnose and manage non-malarial fevers. Educating caregivers on signs and symptoms of febrile illness, as well as the introduction of additional tests to differentiate between viral and bacterial illness, would be important steps to get children to PHCFs earlier and decrease unnecessary antibiotic prescribing without compromising patient safety. More research is needed to expand an understanding of what would improve fever management in other resource-limited settings with decreasing malaria.

Epithelial cells in nipple aspirate fluid and subsequent breast cancer risk: A historic prospective study

BackgroundPast studies have shown that women with abnormal cytology or epithelial cells in nipple aspirate fluid (NAF) have an increased relative risk (RR) of breast cancer when compared to women from whom NAF was attempted but not obtained (non-yielders). This study analyzed NAF results from a group of women seen in a breast clinic between 1970–1991 (N = 2480). Our analysis presented here is an aggregate of two sub-groups: women with questionnaire data (n = 712) and those with NAF visits beginning in 1988 (n = 238), the year in which cancer case information was uniformly collected in California.MethodsCytological classification was determined for a group of 946 women using the most abnormal epithelial cytology observed in fluid specimens. Breast cancer incidence and mortality status was determined through June 2006 using data from the California Cancer Registry, California Vital Statistics and self-report. We estimated odd ratios (ORs) for breast cancer using logistic regression analysis, adjusting for age. We analyzed breast cancer risk related to abnormality of NAF cytology using non-yielders as the referent group and breast cancer risk related to the presence or absence of epithelial cells in NAF, using non-yielders/fluid without epithelial cells as the referent group.ResultsOverall, 10% (93) of the 946 women developed breast cancer during the follow-up period. Age-adjusted ORs and 95% confidence intervals (C.I.) compared to non-yielders were 1.4 (0.3 to 6.4), 1.7 (0.9 to 3.5), and 2.0 (1.1 to 3.6) for women with fluid without epithelial cells, normal epithelial cells and hyperplasia/atypia, respectively. Comparing the presence or absence of epithelial cells in NAF, women with epithelial cells present in NAF were more likely to develop breast cancer than non-yielders or women with fluid without epithelial cells (RR = 1.9, 1.2 to 3.1).ConclusionThese results support previous findings that 1) women with abnormal epithelial cells in NAF have an increased risk of breast cancer when compared to non-yielders or women with normal epithelial cells in NAF and 2) women with epithelial cells present in NAF have an increased risk of breast cancer when compared to non-yielders or women who had NAF without epithelial cells present.

Impact of introduction of rapid diagnostic tests for malaria on antibiotic prescribing: analysis of observational and randomised studies in public and private healthcare settings

Objectives To examine the impact of use of rapid diagnostic tests for malaria on prescribing of antimicrobials, specifically antibiotics, for acute febrile illness in Africa and Asia. Design Analysisof nine preselected linked and codesigned observational and randomised studies (eight cluster or individually randomised trials and one observational study). Setting Public and private healthcare settings, 2007-13, in Afghanistan, Cameroon, Ghana, Nigeria, Tanzania, and Uganda. Participants 522 480 children and adults with acute febrile illness. Interventions Rapid diagnostic tests for malaria. Main outcome measures Proportions of patients for whom an antibiotic was prescribed in trial groups who had undergone rapid diagnostic testing compared with controls and in patients with negative test results compared with patients with positive results. A secondary aim compared classes of antibiotics prescribed in different settings. Results Antibiotics were prescribed to 127 052/238 797 (53%) patients in control groups and 167 714/283 683 (59%) patients in intervention groups. Antibiotics were prescribed to 40% (35 505/89 719) of patients with a positive test result for malaria and to 69% (39 400/57 080) of those with a negative result. All but one study showed a trend toward more antibiotic prescribing in groups who underwent rapid diagnostic tests. Random effects meta-analysis of the trials showed that the overall risk of antibiotic prescription was 21% higher (95% confidence interval 7% to 36%) in intervention settings. In most intervention settings, patients with negative test results received more antibiotic prescriptions than patients with positive results for all the most commonly used classes: penicillins, trimethoprim-sulfamethoxazole (one exception), tetracyclines, and metronidazole. Conclusions Introduction of rapid diagnostic tests for malaria to reduce unnecessary use of antimalarials—a beneficial public health outcome—could drive up untargeted use of antibiotics. That 69% of patients were prescribed antibiotics when test results were negative probably represents overprescription.This included antibiotics from several classes, including those like metronidazole that are seldom appropriate for febrile illness, across varied clinical, health system, and epidemiological settings. It is often assumed that better disease specific diagnostics will reduce antimicrobial overuse, but they might simply shift it from one antimicrobial class to another. Current global implementation of malaria testing might increase untargeted antibiotic use and must be examined.

Perception of malaria risk in a setting of reduced malaria transmission: a qualitative study in Zanzibar

BackgroundMalaria transmission has declined dramatically in Zanzibar in recent years. Continuing use of preventive measures such as long-lasting insecticidal-treated nets (LLINs), and use of malaria rapid diagnostic tests (RDTs) are essential to prevent malaria resurgence. This study employed qualitative methods to explore community perceptions of malaria risk and adherence to prevention measures in two districts in Zanzibar.MethodsKey informant interviews with 24 primary health care providers and 24 focus group discussions with local residents in Zanzibar districts Wete and Central were conducted during April and May 2012 focusing on perception of malaria risk, current preventive practices used, reasons for using preventive practices and effective strategies for malaria control.ResultsHealth care providers and residents appear to be aware of the decreasing incidence of malaria. Both groups continue the use of malaria preventive practices in this low and seasonal transmission setting. The most important preventive measures identified were LLINs, indoor residual spraying (IRS), and education. Barriers to malaria prevention include: lack of staff at clinics, insufficient number of LLINs distributed, and inadequate malaria education. Reasons for continued use of preventive practices include: fear of malaria returning to high levels, presence of mosquitoes during rainy seasons, and concern about local cases from other villages or imported cases from mainland Tanzania. Mosques, clinics, schools and community meetings were listed as most important sources of education. However, residents express the desire for more education.ConclusionHealth care providers and residents generally reported consistent use of malaria preventive measures. However, maintaining and continuing to reduce malaria transmission will require ongoing education for both health care providers and residents to reinforce the importance of using preventive measures. Successful efforts to reduce malaria in Zanzibar will be jeopardized if residents believe that they are no longer at risk for malaria. In future studies, a year-round evaluation of the perception of malaria risk and use of preventive measures will inform the timing of education and prevention strategies for sustained malaria control.

Prevalence of PCR Detectable Malaria Infection among Febrile Patients with a Negative Plasmodium falciparum Specific Rapid Diagnostic Test in Zanzibar

We screened for malaria in 594 blood samples from febrile patients who tested negative by a Plasmodium falciparum-specific histidine-rich protein-2-based rapid diagnostic test at 12 health facilities in Zanzibar districts North A and Micheweni, from May to August 2010. Screening was with microscopy, polymerase chain reaction (PCR) targeting the cytochrome b gene (cytbPCR) of the four major human malaria species, and quantitative PCR (qPCR). The prevalence of cytbPCR-detectable malaria infection was 2% (12 of 594), including 8 P. falciparum, 3 Plasmodium malariae, and 1 Plasmodium vivax infections. Microscopy identified 4 of 8 P. falciparum infections. Parasite density as estimated by microscopy or qPCR was > 4,000 parasites/μL in 5 of 8 cytbPCR-detectable P. falciparum infections. The infections that were missed by the rapid diagnostic test represent a particular challenge in malaria elimination settings and highlight the need for more sensitive point-of-care diagnostic tools to improve case detection of all human malaria species in febrile patients.

Acute Uncomplicated Febrile Illness in Children Aged 2-59 months in Zanzibar – Aetiologies, Antibiotic Treatment and Outcome

Background Despite the fact that a large proportion of children with fever in Africa present at primary health care facilities, few studies have been designed to specifically study the causes of uncomplicated childhood febrile illness at this level of care, especially in areas like Zanzibar that has recently undergone a dramatic change from high to low malaria transmission. Methods We prospectively studied the aetiology of febrile illness in 677 children aged 2–59 months with acute uncomplicated fever managed by IMCI (Integrated Management of Childhood Illness) guidelines in Zanzibar, using point-of-care tests, urine culture, blood-PCR, chest X-ray (CXR) of IMCI-pneumonia classified patients, and multiple quantitative (q)PCR investigations of nasopharyngeal (NPH) (all patients) and rectal (GE) swabs (diarrhoea patients). For comparison, we also performed NPH and GE qPCR analyses in 167 healthy community controls. Final fever diagnoses were retrospectively established based on all clinical and laboratory data. Clinical outcome was assessed during a 14-day follow-up. The utility of IMCI for identifying infections presumed to require antibiotics was evaluated. Findings NPH-qPCR and GE-qPCR detected ≥1 pathogen in 657/672 (98%) and 153/164 (93%) of patients and 158/166 (95%) and 144/165 (87%) of controls, respectively. Overall, 57% (387/677) had IMCI-pneumonia, but only 12% (42/342) had CXR-confirmed pneumonia. Two patients were positive for Plasmodium falciparum. Respiratory syncytial virus (24.5%), influenza A/B (22.3%), rhinovirus (10.5%) and group-A streptococci (6.4%), CXR-confirmed pneumonia (6.2%), Shigella (4.3%) were the most common viral and bacterial fever diagnoses, respectively. Blood-PCR conducted in a sub-group of patients (n = 83) without defined fever diagnosis was negative for rickettsiae, chikungunya, dengue, Rift Valley fever and West Nile viruses. Antibiotics were prescribed to 500 (74%) patients, but only 152 (22%) had an infection retrospectively considered to require antibiotics. Clinical outcome was generally good. However, two children died. Only 68 (11%) patients remained febrile on day 3 and three of them had verified fever on day 14. An additional 29 (4.5%) children had fever relapse on day 14. Regression analysis determined C-reactive Protein (CRP) as the only independent variable significantly associated with CXR-confirmed pneumonia. Conclusions This is the first study on uncomplicated febrile illness in African children that both applied a comprehensive laboratory panel and a healthy control group. A majority of patients had viral respiratory tract infection. Pathogens were frequently detected by qPCR also in asymptomatic children, demonstrating the importance of incorporating controls in fever aetiology studies. The precision of IMCI for identifying infections requiring antibiotics was low.

Epstein-Barr virus is seldom found in mammary epithelium of breast cancer tissue using in situ molecular methods

Epstein–Barr virus (EBV) has been proposed as a possible etiological agent of breast cancer based on 21 reports of EBV in malignant breast tissues. Most of these studies used standard and nested solution polymerase chain reaction (PCR) techniques, both disadvantaged by susceptibility to contamination from laboratory EBV, and the inability to localize the signal to a specific cell type. To avoid these issues, we used in situ molecular methods of viral detection to reassess the frequency of EBV in malignant breast tissue. We used a commercial in situ hybridization (ISH) system with an EBER genome target, and a non-commercial in situ PCR (IS-PCR) method using primers specific for the BamH1 region. The assays were performed on malignant breast tissue sections from 70 breast cancer patients at the M. D. Anderson Cancer Center, Houston, TX. EBV was found in mammary epithelial cells, the cell type from which most breast cancers arise, in 2/70 (2.9%) of specimens using IS-PCR and in none of the specimens using ISH. Based on these findings that EBV was present in human mammary epithelial cells so infrequently, it is unlikely to play a causative role in most types of breast cancer.