Nasal localization of a Pseudoterranova decipiens larva in a Danish patient with suspected allergic rhinitis.

Pseudoterranoviasis is a zoonotic disease caused by nematode larvae of species within the genus Pseudoterranova (seal worm, cod worm). Most infections are gastrointestinal, oesophageal or pharyngeal, but here we report a nasal infection. A 33-year-old patient suffering from rhinitis for 1.5 years recovered a worm larva from the nose. Diagnosis was performed by morphological and molecular characterization, showing the causative agent to be a third-stage larva of Pseudoterranova decipiens (sensu stricto). Various infection routes are discussed.

Risk factors for surgical site infections in abdominal surgeries in Ghana: emphasis on the impact of operating rooms door openings.

Major surgery carried out in low- and middle-income countries is associated with a high risk of surgical site infections (SSI), but knowledge is limited regarding contributory factors to such infections. This study explores factors related to patients developing an SSI in a teaching hospital in Ghana. A prospective cohort study of patients undergoing abdominal surgical procedures was conducted at Korle Bu Teaching Hospital. Patient characteristics, procedures and environmental characteristics were recorded. A 30-day daily surveillance was used to diagnose SSI, and Poisson regression analysis was used to test for association of SSI and risk factors; survival was determined by proportional hazard regression methods. We included 358 patients of which 58 (16.2%; 95% CI 12.7-20.4%) developed an SSI. The median number of door openings during an operation was 79, with 81% being unnecessary. Door openings greater than 100 during an operation (P = 0.028) significantly increased a patient's risk of developing an SSI. Such patients tended to have an elevated mortality risk (hazard ratio 2.67; 95% CI 0.75-9.45, P = 0.128). We conclude that changing behaviour and practices in operating rooms is a key strategy to reduce SSI risk.

Surveillance of surgical site infection in a teaching hospital in Ghana: a prospective cohort study.

BACKGROUND: Surveillance systems for surgical site infections (SSIs), as a measure of patient safety, help health institutions devise strategies to reduce or prevent them. No surveillance systems exist to monitor SSIs in Ghana. AIM: To establish a system for monitoring trends and detecting outbreaks in order to create awareness of and control SSIs. METHODS: An active 30-day surveillance was undertaken at the general surgical unit of the Korle Bu Teaching Hospital, from July 1st, 2017 to December 31st, 2018 to identify SSI. It involved a daily inpatient surveillance of patients who had had a surgical procedure, followed by post-discharge surveillance by means of a healthcare personnel-based survey and a patient-based telephone survey. We supplied quarterly feedback of results to surgeons. FINDINGS: Among the 3267 patients included, 331 were identified with an SSI, a 10% incidence risk. Patients who acquired an SSI experienced increased morbidity including nine extra days in hospital and an adjusted relative mortality risk of 2.3 (95% confidence interval: 1.3 - 4.1; P=0.006) compared to patients without SSI. Forty-nine per cent (161/331) of SSIs were diagnosed post discharge using the healthcare personnel-based survey. The patient-based telephone survey contributed 12 additional cases. SSI incidence risk decreased from 12.8% to 7.5% during the study period. CONCLUSION: Post-discharge surveillance is feasible using existing healthcare personnel, and the results highlight the high risk and burden of SSIs in Ghana. A surveillance system with feedback for monitoring SSIs may contribute to reducing SSIs; however, firm conclusions regarding the impact need longer observation time.

Metronidazole-sensitive organisms in children with severe acute malnutrition: an evaluation of the indication for empiric metronidazole treatment.

OBJECTIVES: Children with severe acute malnutrition (SAM) are treated with empiric amoxicillin or penicillin and gentamicin because of the high risk of severe infections. Experts have suggested, based on available evidence, adding metronidazole to cover anaerobic bacteraemia and diarrhoea caused by Giardia duodenalis or Clostridium difficile. The objective of this study was to assess the importance of these infections in children with SAM. METHODS: Children from 6 months to 15 years with SAM were enrolled and followed clinically. Aerobic and, when patient weight permitted, anaerobic blood cultures were done using Bactec® system, and isolates identified with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Stool samples were tested for C. difficile, G. duodenalis and Entamoeba histolytica by PCR. RESULTS: A total of 334 children were enrolled and 174 out of 331 (53%) for which data on this was available had diarrhoea. Of 273 patients tested by blood culture, 11 had bacteraemia (4.0%, 95% CI 2.3-7.1%) but none with strict anaerobic bacteria (0/153, 95% CI 0-2.4%). There was no difference in the prevalence of C. difficile between children with (5/128, 4%) and without (7/87, 8%) diarrhoea (OR 0.47, 95% CI 0.14-1.53), and no difference in the prevalence of Giardia between these groups (78/138, 60% vs. 46/87, 53%; OR 1.34, 95% CI 0.77-2.32). Children with C. difficile had higher mortality than those without this infection (3/11, 27%, vs. 7/186, 4%; OR 43, 95% CI 3.9-483). CONCLUSION: Our results do not provide support for empiric metronidazole to cover for anaerobic bacteraemia. Trials evaluating the effect of empiric treatment and its effect on G. duodenalis and C. difficile are warranted.

Genetic relationship between bacteria isolated from intraoperative air samples and surgical site infections at a major teaching hospital in Ghana.

BACKGROUND: In low- and middle-income countries (LMICs) the rate of surgical site infections (SSI) is high, leading to negative patient outcomes and excess healthcare costs. A causal relationship between airborne bacteria in the operating room and SSI has not been established, at a molecular or genetic level. We studied the relationship between intraoperative airborne bacteria and bacteria causing SSI in an LMIC. METHODS: Active air sampling using a portable impactor was performed during clean or clean-contaminated elective surgical procedures. Active patient follow-up consisting of phone calls and clinical examinations was performed 3, 14 and 30 days after surgery. Bacterial isolates recovered from SSI and air samples were compared by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) identification, ribotyping, whole genome sequencing (WGS), and metagenomic analysis. RESULTS: Of 128 included patients, 116 (91%) completed follow-up and 11 (9%) developed SSI. Known pathogenic bacteria were isolated from intraoperative air samples in all cases with SSI. A match between air and SSI isolates was found by MALDI-TOF in eight cases. Matching ribotypes were found in six cases and in one case both WGS and metagenomic analysis showed identity between air- and SSI-isolates. CONCLUSION: The study showed high levels of intraoperative airborne bacteria, an SSI-rate of 9% and a genetic link between intraoperative airborne bacteria and bacteria isolated from SSIs. This indicates the need for awareness of intraoperative air quality in LMICs.

Traffic flow and microbial air contamination in operating rooms at a major teaching hospital in Ghana.

BACKGROUND: Current literature examining the relationship between door-opening rate, number of people present, and microbial air contamination in the operating room is limited. Studies are especially needed from low- and middle-income countries, where the risk of surgical site infections is high. AIM: To assess microbial air contamination in operating rooms at a Ghanaian teaching hospital and the association with door-openings and number of people present. Moreover, we aimed to document reasons for door-opening. METHODS: We conducted active air-sampling using an MAS 100® portable impactor during 124 clean or clean-contaminated elective surgical procedures. The number of people present, door-opening rate and the reasons for each door-opening were recorded by direct observation using pretested structured observation forms. FINDINGS: During surgery, the mean number of colony-forming units (cfu) was 328 cfu/m3 air, and 429 (84%) of 510 samples exceeded a recommended level of 180 cfu/m3. Of 6717 door-openings recorded, 77% were considered unnecessary. Levels of cfu/m3 were strongly correlated with the number of people present (P = 0.001) and with the number of door-openings/h (P = 0.02). In empty operating rooms, the mean cfu count was 39 cfu/m3 after 1 h of uninterrupted ventilation and 52 (51%) of 102 samples exceeded a recommended level of 35 cfu/m3. CONCLUSION: The study revealed high values of intraoperative airborne cfu exceeding recommended levels. Minimizing the number of door-openings and people present during surgery could be an effective strategy to reduce microbial air contamination in low- and middle-income settings.

New filtration system for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts.

AIMS: To develop a filtration unit for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts ((oo-)cysts) in drinking water. METHODS AND RESULTS: This unit utilizes a metallic filter and an ultrasound transducer for eluting (oo-)cysts, with a fixed retentate backwash volume; approx. 400 µl. Changes in the viability was evaluated by seeding wild type (oo-)cysts (1 × 10(4)) followed by sonication for 5, 10, 20 or 40 s (five replicates for each period). Flow cytometry analysis showed negligible increase in the mortality of (oo-)cysts exposed to 5-10 s of sonication. Recovery rate was assessed by seeding ColorSeed(™) (10 replicates) into the filter unit followed by air backwash to a glass slide and counting of (oo-)cysts by epifluorescent microscopy. High recovery rates (mean ± SD) were found: 84·9% ± 4·8 for Giardia cysts and 70% ± 6·5 for Cryptosporidium oocysts. DNA of seeded wild type (oo-)cysts (1 × 10(2); 10 replicates) was successfully amplified using real-time PCR. CONCLUSIONS: The use of a metallic filter, sonication and 'air backwash' were key factors for creating a highly efficient system for recovery of apparently undamaged protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: This reagent-less system can be used for monitoring of parasite contamination in drinking water.

Implementation of minimally invasive and objective humane endpoints in the study of murine Plasmodium infections.

SUMMARY Defining appropriate and objective endpoints for animal research can be difficult. Previously we evaluated and implemented a body temperature (BT) of <32 °C as an endpoint for experimental cerebral malaria (ECM) and were interested in a similar endpoint for a model of severe malarial anaemia (SMA). Furthermore, we investigate the potential of a minimally invasive, non-contact infrared thermometer for repeated BT measurement. ECM was induced with Plasmodium berghei ANKA infection in C57Bl/6 mice. SMA was induced with Plasmodium chabaudi AS infection in A/J mice. Our previous published endpoint was applied in ECM and 30 °C was pre-determined as the lowest permitted limit for termination in SMA according to consultation with the Danish Animal Inspectorate. Infrared thermometer was compared with the rectal probe after cervical dislocation, ECM and SMA. Linear regression analysis of rectal versus infrared thermometry: cervical dislocation: Pearson R = 0·99, R 2 = 0·98, slope = 1·01, y-intercept = 0·55; ECM: 0·99, 0·98, 1·06, -2·4; and SMA: 0·98, 0·97, 1·14, -5·6. Implementation of the 30 °C endpoint captured all lethal infections. However, some animals with BT below 30 °C were not deemed clinically moribund. This study supports repeated measurement infrared thermometry. A humane endpoint of 30 °C was sensitive in capturing terminal animals but might overestimate lethality in this SMA model.

In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei.

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes. In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.

Parents' perceptions, attitudes and acceptability of treatment of childhood malaria with artemisinin combination therapies in ghana.

BACKGROUND: There is little information on sociocultural and contextual factors that may influence attitudes of patients to new treatments, such as artemisinin combination therapies (ACT). METHODS: Semi-structured questionnaires and focus group discussions were used to assess views of parents of children with uncomplicated malaria treated with ACT in a low socio-economic area in Accra, Ghana. RESULTS: The majority of parents reported a favourable experience, in terms of perceived i) rapidity of symptom resolution, compared to their previous experience of other therapies for childhood malaria, or ii) when their experience was compared that of parents of children treated with monotherapy. The parents of children treated with ACT were more willing to pay for the treatment, or adhere to the full treatment course. The explanations given for adherence were consistent with conventional biomedical explanations. Although care-seeking practices for childhood malaria were considered appropriate, perceived or real barriers to accessible health care were also important factors in the decision to seek treatment. Household dynamics and perceived inequities at the care-provider-patient interface were identified as having potential negative impact on care-seeking practices and adherence. CONCLUSIONS: Health education messages aimed at improving the response to childhood febrile illness should include other strategic stakeholders, such as decision-makers at the household level. The effectiveness and implementation success of the ACT policy could be enhanced by highlighting and reinforcing messages intrinsic to these regimens. Integrating the views of caretakers during the clinical encounter was validated as an empowerment tool that could aid in the appropriate responses to childhood illness.

Circulating epstein-barr virus in children living in malaria-endemic areas.

Children living in malaria-endemic regions have high incidence of Burkitt's lymphoma (BL), the aetiology of which involves Plasmodium falciparum malaria and Epstein-Barr virus (EBV) infections. Acute malarial infection impairs the EBV-specific immune responses with the consequent increase in the number of EBV-carrying B cells in the circulation. To further understand the potential influence of malarial infection on the EBV persistence in children living in malaria-endemic areas, we studied the occurrence and quantified cell-free EBV-DNA in plasma from 73 Ghanaian children with and without acute malarial infection. Viral DNA was detected in 40% of the samples (47% in the malaria-infected and 34% in the nonmalaria group) but was absent in plasma from Ghanaian adults and healthy Italian children. These findings provide evidence that viral reactivation is common among children living in malaria-endemic areas, and may contribute to the increased risk for endemic BL. The data also suggest that the epidemiology of EBV infection and persistence varies in different areas of the world.

Cytokine production and apoptosis among T cells from patients under treatment for Plasmodium falciparum malaria.

Available evidence suggests that Plasmodium falciparum malaria causes activation and reallocation of T cells, and that these in vivo primed cells re-emerge into the periphery following drug therapy. Here we have examined the cytokine production capacity and susceptibility to programmed cell death of peripheral T cells during and after the period of antimalarial treatment. A high proportion of peripheral CD3+ cells had an activated phenotype at and shortly after time of admission (day 0) and initiation of therapy. This activation peaked around day 2, and at this time-point peripheral T cells from the patients could be induced to produce cytokines at conditions of limited cytokine response in cells from healthy control donors. Activated CD8hi and TCR-gammadelta+ cells were the primary IFN-gamma producers, whereas CD4+ cells constituted an important source of TNF-alpha. The proportion of apoptotic T cells was elevated at admission and peaked 2 days later, while susceptibility to activation-induced cell death in vitro remained increased for at least 1 week after admission. Taken together, the data are consistent with the concept of malaria-induced reallocation of activated T cells to sites of inflammation, followed by their release back into the peripheral blood where they undergo apoptotic death to re-establish immunological homeostasis as inflammation subsides. However, the high proportion of pre-apoptotic cells from the time of admission suggests that apoptosis also contributes to the low frequency and number of T cells in the peripheral circulation during active disease.

Complement binding to erythrocytes is associated with macrophage activation and reduced haemoglobin in Plasmodium falciparum malaria.

We have examined IgG and complement factor C3d deposition on erythrocytes by means of the direct Coombs' test (DAT) and looked for an association with the anaemia seen in falciparum malaria in children living in an area of hyperendemic malaria transmission (in Ghana). In one study (in 1997), 53 out of 199 patients had a positive DAT. Of these, 45 samples reacted with anti-C3d antibodies, 2 with anti-IgG and 6 with both reagents. There were significantly lower haemoglobin (Hb)-levels and higher prevalence of spleen enlargement in DAT-positive than in DAT-negative patients. Hb-levels were independently associated with DAT and age. This initial study was designed to investigate the role of intravascular haemolysis (IVH), but we found no association between IVH and either DAT result or anaemia. Because of the risk of selection bias we repeated the study using consecutive enrollment of malaria patients and were able to confirm the results in a total of 49 DAT-positive and 183 DAT-negative patients. This second study (in 1998) was designed to look at the importance of erythrophagocytosis through measurement of plasma neopterin levels and total nitrite and nitrate as markers of NO-release. Both parameters were significantly higher in DAT-positive than in DAT-negative patients (P < 0.001), indicating that complement binding to erythrocytes was associated with macrophage activation. Plasma levels of haptoglobin, interleukin-10 and tumour necrosis factor-alpha did not vary between the groups. The studies support the role of complement activation and erythrophagocytosis in the pathogenesis of anaemia in falciparum malaria in African children.

Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity.

Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinical P. falciparum malaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of different H-2 haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria.

Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of PfEMP1 by enzyme-linked immunosorbent assay and antibodies to variant molecules on erythrocytes infected with field isolates of P. falciparum by flow cytometry. Based on close clinical monitoring, the children were grouped into those who did (susceptible) and those who did not (protected) have malaria during the season. The prevalences of antibodies to both the conserved PfEMP1 peptide and the variant epitopes were greater than 50%, and the levels of immunoglobulin G (IgG) correlated with age. The levels of antibodies to both the conserved peptide and the variant epitopes were higher in protected than in susceptible children. After correcting for the effect of age, the levels of IgG to variant antigens on a Sudanese and a Ghanaian parasite isolate remained significantly higher in protected than in susceptible children. Thus, the levels of IgG to variant antigens expressed on the surface of infected erythrocytes correlated with protection from clinical malaria. In contrast, the levels of IgG to a peptide derived from a conserved part of PfEMP1 did not correlate with protection from malaria.

Perturbation and proinflammatory type activation of V delta 1(+) gamma delta T cells in African children with Plasmodium falciparum malaria.

gamma delta T cells have variously been implicated in the protection against, and the pathogenesis of, malaria, but few studies have examined the gamma delta T-cell response to malaria in African children, who suffer the large majority of malaria-associated morbidity and mortality. This is unfortunate, since available data suggest that simple extrapolation of conclusions drawn from studies of nonimmune adults ex vivo and in vitro is not always possible. Here we show that both the frequencies and the absolute numbers of gamma delta T cells are transiently increased following treatment of Plasmodium falciparum malaria in Ghanaian children and they can constitute 30 to 50% of all T cells shortly after initiation of antimalarial chemotherapy. The bulk of the gamma delta T cells involved in this perturbation expressed V delta 1 and had a highly activated phenotype. Analysis of the T-cell receptors (TCR) of the V delta 1(+) cell population at the peak of their increase showed that all expressed V gamma chains were used, and CDR3 length polymorphism indicated that the expanded V delta 1 population was highly polyclonal. A very high proportion of the V delta 1(+) T cells produced gamma interferon, while fewer V delta 1(+) cells than the average proportion of all CD3(+) cells produced tumor necrosis factor alpha. No interleukin 10 production was detected among TCR-gamma delta(+) cells in general or V delta 1(+) cells in particular. Taken together, our data point to an immunoregulatory role of the expanded V delta 1(+) T-cell population in this group of semi-immune P. falciparum malaria patients.

Haptoglobin 1-1 is associated with susceptibility to severe Plasmodium falciparum malaria.

The haptoglobin (Hp) phenotypes were determined by polyacrylamide-gel electrophoresis in plasma samples obtained in 1997 from 113 Plasmodium falciparum malaria patients (aged 1-12 years) with strictly defined cerebral malaria, severe malarial anaemia, or uncomplicated malaria and 42 age-matched healthy controls from the same area (coastal Ghana). Hp1-1 was significantly more prevalent among the patients (43%) than among healthy controls (7.1%), whereas Hp2-1 and Hp2-2 were underrepresented among the patients (11% and 2%, respectively) compared to the control donors (33% and 14%, respectively). No significant difference in frequency of Hp0 was observed between patients and controls. Among the malaria patients, the Hp1-1 phenotype was significantly more prevalent among patients with the complications of cerebral malaria and severe anaemia compared to patients with uncomplicated disease, whereas the reverse was seen with respect to Hp2-1 and Hp2-2. Our data suggest that the Hp1-1 phenotype is associated with susceptibility to P. falciparum malaria in general, and to the development of severe disease in particular.

High frequency of circulating gamma delta T cells with dominance of the v(delta)1 subset in a healthy population.

TCR gamma delta(+) cells constitute <5% of all circulating T cells in healthy, adult Caucasians, and V(delta)1(+) cells constitute a minority of these cells. In contrast to TCR alpha beta(+) cells, their repertoire is selected extrathymically by environmental antigens. Although increased frequencies of V(delta)1(+) cells are found in several diseases, their function remains obscure. Here we show that the frequency of peripheral blood gamma delta T cells in healthy West Africans is about twice that of Caucasians, mainly due to a 5-fold increase in V(delta)1(+) cells, which is consequently the dominant subset. No age dependency of V(delta)1 frequencies was identified and the V(delta)1(+) cells in the African donors did not show preferential V(gamma) chain usage. Analysis of the CDR3 region size did not reveal any particular skewing of the V(delta)1 repertoire, although oligoclonality was more pronounced in adults compared to children. The proportions of CD8(+), CD38(+) and CD45RA(hi)CD45RO(-) cells within the V(delta)1(+) subset were higher in the African than in the European donors, without obvious differences in expression of activation markers. No significant correlations between levels of V(delta)1(+) cells and environmental antigens or immunological parameters were identified. Taken together, the evidence argues against a CDR3-restricted, antigen-driven expansion of V(delta)1(+) cells in the African study population. Our study shows that high frequencies of TCR gamma delta(+) cells with dominance of the V(delta)1(+) subset can occur at the population level in healthy people, raising questions about the physiological role of V(delta)1(+) T cells in the function and regulation of the immune system.