Evidence of Hantavirus Infection among Bats in Brazil

Hantaviruses are zoonotic viruses harbored by rodents, bats, and shrews. At present, only rodent-borne hantaviruses are associated with severe illness in humans. New species of hantaviruses have been recently identified in bats and shrews greatly expanding the potential reservoirs and ranges of these viruses. Brazil has one of the highest incidences of hantavirus cardiopulmonary syndrome in South America, hence it is critical to know what is the prevalence of hantaviruses in Brazil. Although much is known about rodent reservoirs, little is known regarding bats. We captured 270 bats from February 2012 to April 2014. Serum was screened for the presence of antibodies against a recombinant nucleoprotein (rN) of Araraquara virus (ARAQV). The prevalence of antibody to hantavirus was 9/53 with an overall seroprevalence of 17%. Previous studies have shown only insectivorous bats to harbor hantavirus; however, in our study, of the nine seropositive bats, five were frugivorous, one was carnivorous, and three were sanguivorous phyllostomid bats.Hantaviruses (family Bunyaviridae) are present throughout the globe in rodents, bats, and shrews.¹ Humans exposed to rodent excreta from hantaviral reservoirs may develop life-threatening diseases. However, none of the other reservoirs are associated with human illness presently.^1,2 Bats (order Chiroptera) are known to harbor a broad diversity of emerging zoonotic pathogens.² Their ability to fly and social behavior favors maintenance, evolution, and spread of pathogens.^1,2 The prevailing hypothesis has been that hantaviruses have coevolved with their rodent reservoirs over millions of years.^1,3 With the recognition of new species of hantavirus in bats in Africa and Asia,⁴ Guo and others⁵ hypothesized that hantaviruses originated primarily in bats and then spilled over into rodents and shrews, but it seems that shrews are the original hosts from which the viruses jumped into both rodents and bats.³ To determine if New World bats in Brazil may harbor hantaviruses, we screened bat sera for antibodies that react against the recombinant nucleoprotein (rN) of Araraquara hantavirus (ARAQV).Bats were collected at five ecologically distinct sites in the northeast region of São Paulo state (sites 1–3) and north region of Minas Gerais state (sites 4 and 5), southeastern Brazil (Figure 1 and 9 and one specimen per species by trap-night was anesthetized to collect blood by cardiac puncture; blood samples were stored in cryovials and flash-frozen in liquid nitrogen. At sites 4 and 5, five specimens per trap-night were randomly selected for blood collection. All bats were handled and sampled according to Sikes and others¹⁰ guidelines. This research project, along with its procedures and protocols, is in accordance with Brazilian environment and wildlife protection laws and regulations, and have been approved by the Chico Mendes Institute of Biodiversity Conservation (Ministry of Environment, Brasília, Distrito Federal, Brazil.), protocols nos. 19838-1 and 41709-3. It has also been approved by the Ethics Committee for Animal Research of University of São Paulo and Federal University of Minas Gerais (nos. 020/2011 and 333/2013, respectively). From 270 captured bats, 53 were bled for detection of immunoglobulin G (IgG) antibodies to rN-ARAQV by indirect enzyme-linked immunosorbent assay (ELISA) using anti-bat (Bethyl Laboratories, Inc., Montgomery, TX) secondary antibody. This ELISA, as previously described, showed 97.2% sensitivity, 100% specificity, 100% positive predictive value, and 98.1% negative predictive value when compared with an IgG-ELISA using rN antigen of Andes virus, which is the serological test for hantavirus most used in South America.^11,12Open in a separate windowFigure 1.Study areas, highlighting the states of São Paulo and Minas Gerais in southeastern Brazil. The map shows cities where bats have been captured.

Table 1

Trap sites general features⁶

Diabetes: A Contributor to Tuberculosis in Tropical Australia

In countries with a high-burden of tuberculosis (TB), it has been well established that there is an increased incidence of TB among patients with diabetes. However, in countries with a low burden of TB there are conflicting reports. This study aimed to determine if diabetes was associated with TB in patients admitted to a teaching hospital in tropical Australia. A 20-year retrospective study found patients with comorbid diabetes were seven times overrepresented in the TB patient population when compared with the general population. This study demonstrates a strong association between TB and diabetes regardless of TB endemicity.Globally, tuberculosis (TB) is the single leading bacterial cause of death.¹ The increasing emergence of comorbid immune suppressing conditions has accounted for the resurgence in TB.^2,3 Patients with diabetes and prediabetes have an increased risk of bacterial infections and demonstrate a poorer prognosis.^4,5 Current estimates reveal patients with diabetes have on average a 3-fold risk of developing active TB when compared with the general population. In some regions, up to 50% of TB is associated with diabetes.^6,7In low-burden TB countries such an association has not been reported and has been attributed to better glycemic control among all patients with diabetes.^8,9 It has been suggested that the association of TB and diabetes may not be a problem in these countries with a low burden of TB. The prevalence of comorbid TB and diabetes also remains ill-defined in many tropical areas within the Western Pacific region for both low- and high-burden TB countries, warranting further investigations.^1,8,9 The aim of this study was to determine if diabetes was a risk factor associated with TB in a low-burden tropical region by examining medical records and determining if TB patients had evidence of preexisting diabetes.We undertook a 20-year retrospective investigation in a defined group of patients with culture-confirmed TB admitted to a tertiary referral hospital in tropical Australia (#QTHS/HREC/43). In total, 69 patients were identified with TB between 1995 and 2014 and included in the analysis. The χ² test with Yates correction was used to assess whether a significant association existed between diabetes and TB and clinical outcomes.Comorbid TB and diabetes occurred in 23.2% (N = 16) of patients. There was a significant association between TB and diabetes (P < 0.0001) when compared with the general population of the region. Patients with diabetes were seven times overrepresented in the TB patient population (OR = 6.6; 95% CI = 3.788–11.60) in comparison to the general population. Almost half (N = 7; 43.8%) of the diabetic patients were recorded as having poorly controlled diabetes, as assessed by fasting blood glucose (> 6.0 mmol/L) and HbA1c levels (> 6.5%).Overseas-born individuals and indigenous Australians were overrepresented in patients with TB alone and comorbid diabetes when compared with the general population. The majority of overseas-born patients originated from high-risk TB countries namely Papua New Guinea (N = 16), followed by India (N = 3), Burma (N = 2), and Sudan (N = 2). There was a lower proportion of comorbid TB and diabetes compared with solitary TB in patients originating from both high- and low-risk TB countries (high-risk country: 25.0% versus 50.2%; low-risk country: 6.3% versus 6.5%). In contrast, comorbid TB and diabetes was more prevalent than TB in isolation for Australian-born patients (indigenous Australians: 43.8% versus 28.3%; non-indigenous Australians: 25.0% versus 15.1%).Comorbid diabetes was associated with pulmonary TB (N = 14; 87.5%; P = 0.0103; OR = 7.84) rather than extrapulmonary disease (N = 2; 12.5%). Comorbid diabetes was also associated with smear positivity at TB diagnosis (N = 13; 81.3%; P = 0.0084; OR = 6.603) when compared with non-diabetic patients. A higher proportion of patients with comorbid diabetes had pulmonary cavitation (31.3% versus 20.3%), relapsed disease (18.8% versus 15.9%), and drug resistance (12.5% versus 7.2%) when compared with non-diabetic patients; however, these did not reach significance.This patient-based research demonstrated that an association exists between TB and diabetes in a low-burden TB country. The results from this study support the observation that the prevalence of comorbid TB and diabetes may exceed world estimates, even in a tropical area with a low incidence of TB (9^,11 Recent predictions further validate the impact of the growing diabetes pandemic on TB outcomes. It is estimated that up to 7.8 million TB cases and 1.5 million TB deaths could be averted if interventions reduce diabetes by 35% over the next 10 years.¹²

Table 1

Comparison of sociodemographic and disease characteristics in patients with comorbid tuberculosis diabetes from different study populations

Throat Swab Samples for Diagnosis of Q Fever

Oropharyngeal swabs collected from patients with Q fever from France and from febrile patients from Senegal were tested by molecular assays for Coxiella burnetii. One positive result (0.08%) occurred for only one patient with acute Q fever. Throat swabs cannot replace blood serum samples as diagnostic tools for Q fever.Q fever is a worldwide zoonosis and many human infections are caused by Coxiella burnetii.¹ Atypical pneumonia is one of the most commonly recognized forms of acute Q fever. Most cases are clinically asymptomatic or mild, characterized by a nonproductive cough, fever, and minimal auscultatory abnormalities, but some case-patients have acute respiratory distress.¹ Laboratory diagnosis of Q fever is primarily based on serologic testing for phase I and phase II antigens.^1,2Over the past decade, polymerase chain reactions (PCRs) for detection of C. burnetii DNA have been commonly used to test patients for acute infection before appearance of antibodies and to test clinical samples for Q fever endocarditis.¹ In recent studies, throat swabs and sputum have been proposed as potentially useful tools for C. burnetii genotyping and the detection of Q fever.^3–5The objective of our study was to determine the usefulness of oropharyngeal and nasopharyngeal swabs as diagnostic tools for Q fever. We analyzed a large number of throat swab samples from patients with Q fever in France and from febrile patients in areas of high Q fever incidence in Senegal.⁶Oropharyngeal and nasopharyngeal swab samples were obtained from patients with suspected Q fever and from outpatients with Q fever at an infectious disease consulting hospital (Hopital La Timone, Marseille, France). In addition, throat swab samples were obtained from health centers distributed throughout rural Senegal from patients with fever. The national ethics committee of Senegal and the local ethics committee of Mediterranean University, Marseille, France, approved this study.Patients were classified as definitely having Q fever if serologic or PCR results for C. burnetii were positive.⁷ Q fever was suspected in patients coming in contact with newborn animals, placentas, or wool that was contaminated with parturient fluids from infected animals.¹ A four-fold decrease in the phase I IgG and IgA titers and disappearance of phase II IgM was considered as indicating a past infection.⁸ Swabs from Senegal were transferred to Marseille frozen at −80°C on dry ice in sterile conditions.DNA was extracted from swabs by using a QIAamp Tissue Kit (QIAGEN, Hilden, Germany). Extracted DNA was handled under sterile conditions to avoid cross-contamination at −20°C until assayed by PCR. To detect C. burnetii, DNA was used as a template in a described quantitative PCR (qPCR) specific for the IS1111 spacer region and the less sensitive IS30A spacer region.⁹ Results were considered positive when confirmed by both spacers. Two sets of negative controls (DNA from non-infected swab specimens and sterile water) and a positive control (DNA extracted from the supernatant of a culture of C. burnetii L929) were included in each run. The quality of DNA handling and extraction of samples was verified by qPCR for the housekeeping gene encoding beta-actin.¹⁰ Results were considered negative when qPCR for C. burnetii was negative for both spacers and the cycle threshold values of the beta-actin gene were ≤ 30.We tested 602 swabs collected from 198 patients in France (VariableNo. swabs tested (no. patients)No. swabs collected before treatment (no. positive)No. swabs collected before treatment from patients with respiratory symptoms (no. positive)No. swabs collected during/after treatmentAcute Q fever171 (44)31 (1)22 (1)140Q fever endocarditis234 (54)2714205Pregnant with Q fever2 (1)002Past Q fever infection152 (56)00152Q fever suspicion43 (43)43NP0Febrile patients from Senegal667 (667)667NP0Total1,269 (865)768NP501Open in a separate window^*NP = not provided.For 31 patients with acute Q fever and 27 patients with Q fever endocarditis, a swab sample was collected before the beginning of doxycycline therapy, whereas the pregnant woman was already receiving treatment at the time the swab samples were collected. Among the patients who were not receiving antibiotic therapy, 22 patients with acute Q fever and 14 patients with Q fever endocarditis had respiratory symptoms, including cough, influenza-like symptoms, or radiographic results compatible with atypical pneumonia at the time of sample collection.Only one throat swab was positive for C. burnetii. This sample was from a 49-year-old woman with fever, atypical pneumonia, non-productive cough, and hepatitis, and who reported excessive use of alcohol. The PCR analysis of a throat swab was positive for C. burnetii, and serologic analysis confirmed acute Q fever (IgM titer = 1:50). The PCR analysis of a blood sample taken the same day was also positive for C. burnetii. In addition, in Senegal, we collected 667 swabs from 667 febrile patients, and all were negative for C. burnetii.In this study on throat swabs, we used molecular assays for the detection of C. burnetii in patients with Q fever, and a positive result was obtained for only one patient with acute Q fever. Our qPCR for the detection of C. burnetii was sensitive and versatile and has been evaluated,^6,9,11 and the quality of DNA extraction was verified for all samples.¹⁰ Our qPCR specific for the IS1111 spacer region could detect 10² bacteria/mL.⁹In addition, as a part of a study on the prevalence of Q fever in western Africa, we tested swab samples from febrile persons from areas with high Q fever incidence in Senegal.^6,11 High incidence rates of Q fever among febrile patients were reported in the villages of Dielmo and Ndiop (73 cases/100,000 and 223 cases/100,000 person-years, respectively). These values were much higher than the annual incidence reported in France (2.5 cases/100,000 person-years).^6,12Throat swabs are the traditionally preferred sample collection method for the detection of Mycoplasma pneumonia pneumonia.¹³ However, throat swabs were proposed to be also useful for the detection of other atypical agents of pneumonia, including C. burnetii.⁵ In a study based on a nested PCR specific for the com1 gene encoding a 27-kD outer membrane protein of C. burnetii in Japan, most patients with acute Q fever had a throat swab or sputum positive for C. burnetii, and the authors proposed that serum and respiratory samples should be preferable for the PCR-based screening of patients with acute Q fever.⁵ However, suicide PCR cannot be used as a routine tool for the diagnosis of Q fever because it is performed with single-use primers specific for single-use gene fragments and has not been confirmed in laboratory studies with positive controls to rule out contamination.¹⁴ Moreover, in the Netherlands, patient throat swabs have been commonly used as samples for C. burnetii genotyping.^3,4 However, based on the low number of positive results obtained from our series of Q fever patients, we do not believe that throat swabs can replace blood serum samples as diagnostic tools for Q fever.⁷     

Molecular-Based Assay for Simultaneous Detection of Four Plasmodium spp. and Wuchereria bancrofti Infections

Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)–based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction–fluorescent microsphere assay (LDR-FMA), we developed a multiplex assay that has the capability to simultaneously detect all four Plasmodium spp. and W. bancrofti infections in blood samples. Compared with microfilarial positivity in the blood, the LDR-FMA assay is highly concordant (91%), sensitive (86%), and specific (94%), and has high reproducibility for Plasmodium spp. (85–93%) and W. bancrofti (90%) diagnoses. The development of this assay for the simultaneous diagnosis of multiple parasitic infections enables efficient screening of large numbers of human blood and mosquito samples from co-endemic areas.Globally, malaria and lymphatic filariasis are the most threatening of the mosquito-transmitted parasitic diseases.¹ Among the three parasites that cause lymphatic filariasis, Wuchereria bancrofti, Brugia malayi, and B. timori, W. bancrofti is the most widely distributed and is responsible for 90% of lymphatic filariasis infections (bancroftian filariasis) worldwide.² Malaria and bancroftian filariasis are co-endemic in many tropical and sub-tropical regions, such as Southeast Asia, including the western Pacific, Africa, and Central and South America, and are transmitted by a number of common vector species.^3,4 Thus, co-infections with malaria and bancroftian parasites in humans^5–7 and mosquitoes^7,8 are found in these regions.Because of their significant impact on public health, global campaigns with a variety of approaches have been launched for the control/elimination of these diseases.^9,10 These approaches range from the treatment of clinical patients to the control of disease transmission by preventative chemotherapy and vector control.^9,10 However, challenges lie ahead for the success of these control/elimination programs without thoughtful and appropriate use of highly sensitive and specific diagnostic methods.Parasitologic diagnosis of malaria and bancroftian filariasis is typically made by microscopic examination of stained blood smears or membrane filtrates.^5–7 In addition to microscopic detection of microfilariae, detection of circulating filarial antigen(s) by enzyme-linked immunosorbent assay and immunochromatographic test are other commonly used methods to diagnose bancroftian filariasis.^11–13 A number of polymerase chain reaction (PCR)–based assays are available to separately detect malaria^14,15 and bancroftian filariasis^16–19 parasites. However, only two assays are available to detect these parasite species simultaneously: a multiplex PCR assay for detection of Plasmodium falciparum and W. bancrofti in humans,²⁰ and a real-time multiplex quantitative PCR assay for detection of P. falciparum and W. bancrofti, or P. vivax and W. bancrofti in mosquitoes.²¹Malaria is endemic at altitudes below 1,300–1,600 meters in Papua New Guinea and is the leading cause of illness and death in this country.²² Four major parasite species, P. falciparum, P. vivax, P. malariae, and P. ovale, are transmitted in Papua New Guinea and mixed-species infections are common.^23,24 Recently, we developed a 96-well format, post-PCR ligation detection reaction–fluorescent microsphere assay (LDR-FMA) for multiplex detection of the four major Plasmodium spp,²⁵ and validated its utility in diverse epidemiologic settings.^26,27 Bancroftian filariasis is also endemic in several areas in Papua New Guinea and is a major cause of chronic and acute morbidity.²⁸We have been using the density of microfilariae in blood and an enzyme-linked immunosorbent assay (detection of Og4C3 antigen and anti-Bm14 IgG4) as measures of W. bancrofti infection in our ongoing lymphatic filariasis–related epidemiologic studies.^29–31 However, with decreasing prevalence of W. bancrofti infections, lower microfilaremia, and increasing importance of xenodiagnosis of infection in mosquitoes because of the anticipated success of filariasis elimination programs, DNA-based methods may be more efficient for performing the population-level diagnostic surveillance. Expanding our existing post-PCR LDR-FMA assay, we report the development of a multiplex assay that has the capability to simultaneously detect P. falciparum, P. vivax, P. malariae, and P. ovale, and W. bancrofti infections with high sensitivity and specificity in blood samples.The study was performed according to protocols approved by Institutional Review Boards of University Hospitals Case Medical Center (Protocol 08-05-13) and the Papua New Guinea Institute of Medical Research (Protocol 07-16). Further approval was obtained from the Papua New Guinea Medical Research Advisory Committee (Protocol 6.09). Informed consent was obtained from all study participants at the time of enrollment.This new assay involves a multiplex PCR to amplify genomic regions from Plasmodium spp. (small subunit ribosomal RNA gene fragment)²⁵ and W. bancrofti (long DNA repeat region),¹⁸ followed by a multiplex LDR-FMA to detect P. falciparum, P. vivax, P. malariae, and P. ovale,²⁵ and W. bancrofti in a sequence-specific manner. The PCR reagents and conditions for Plasmodium spp. amplification have been described.^24,25For the multiplex PCR, we evaluated the dNTP concentrations (dATP, dTTP, dGTP, and dCTP) from 200 μM to 800 μM to ensure nucleotide availability for the amplification of both Plasmodium spp. and W. bancrofti genomic regions, and added 0.12 μM of each of W. bancrofti UP (5¢-GATGGTGTATAATAGCAGCA-3¢) and W. bancrofti DN (5¢-GTCATTTATTTCTCCGTCGACTGTC-3¢) amplification primers to the PCR master mixture. The dNTP concentration that performed with consistently high efficiency was 400 μM. The PCR products were subjected to electrophoresis on agarose gels to visualize distinct Plasmodium spp. (491–500 basepairs)²⁵ and W. bancrofti (174 basepairs) amplicons. The PCR products were then subjected to LDR-FMA as described,²⁵ with minor modifications that included use of LDR primers: a W. bancrofti-specific primer (tacactttatcaaatcttacaatcTATATCTGCCCATAGAAATAACTA [sequence in lower case letters represents a 24-basepair oligonucleotide tag]) and a W. bancrofti common primer (Phos 5¢-CGGTGGATCTCTGGTTATCACTCTG-3¢Biotin). In the LDR-fluorescent microsphere hybridization solution containing Plasmodium species-specific fluorescent microspheres,²⁵ we added W. bancrofti -specific fluorescent microsphere #3. Our W. bancrofti PCR and LDR primer sequences are based on the W. bancrofti sequence in GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY297458","term_id":"33415264"}}AY297458).¹⁸To confirm the specificity of our W. bancrofti PCR primers, we amplified an approximately 170-basepair genomic DNA region from one microfilaria isolated from a person in Papua New Guinea and sequenced it. We also sequenced the PCR products from two microfilaria-positive persons from Papua New Guinea whose blood samples were collected as a part of ongoing studies.³² Furthermore, we sequenced the PCR product from five pooled third-stage larvae dissected from mosquitoes that were collected in the Dreikikir District (East Sepik Province, Papua New Guinea), where malaria and lymphatic filariasis are endemic.^24,29–31 All sequences were 100% identical with the W. bancrofti sequence in GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY297458","term_id":"33415264"}}AY297458).The specificity of the assay was further demonstrated by using P. falciparum, P. vivax, P. malariae, and P. ovale, and W. bancrofti genomic DNA samples (Figure 1). Using these genomic DNA samples individually in LDR-FMA reactions containing primers and microspheres for all five species, we found that the assay detected only the parasite species DNA present, and background signals for all other species DNAs were below a median fluorescence intensity of 500. We then performed multiplex PCR LDR-FMA diagnosis to detect Plasmodium spp. and W. bancrofti infections in the blood samples from 517 persons living in the Dreikikir District (East Sepik Province, Papua New Guinea).Open in a separate windowFigure 1.Detection of species-specific DNAs by the five-species multiplex polymerase chain reaction oligonucleotide ligation detection reaction–fluorescent microsphere assay (LDR-FMA). Data represent a summary of five positive-control experiments detecting Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and Wuchereria bancrofti genomic DNAs. Whereas genomic DNAs were added individually, LDR-FMA reactions included oligonucleotide primers and microspheres representing all five species. Numbers in parentheses next to parasite-species designations in the legend ((78), (37), (30), (14), (3)) identify FlexMap™ microspheres (Luminex Corp., Austin, TX). B identifies two blank samples to which no genomic DNAs were added.Using this assay, we found that 443 persons (86%) were infected with at least one of the parasites. Overall infection counts were P. falciparum 346 (67%), P. vivax 176 (34%), P. malariae 116 (22%), P. ovale 35 (7%), and W. bancrofti 175 (34%). Reproducibility of these results was tested for 174 samples (34%). In this analysis, we observed 85–93% concordance for Plasmodium spp. diagnosis and 90% concordance for W. bancrofti diagnosis. Thus, development of this 96-well format assay for simultaneous diagnosis of multiple parasitic infections enables efficient screening of large numbers of samples.Finally, we categorized Plasmodium spp. and W. bancrofti infections into parasite assemblages, which are shown in 24 Most W. bancrofti infections (150 of 175) were observed in various Plasmodium spp. assemblages containing the most prevalent P. falciparum infections (5^–7

Table 1

Prevalence (counts) of not infected and all infections detected by ligation detection reaction–fluorescent microsphere assay^*

Diagnosis of Scrub Typhus

Scrub typhus is transmitted by trombiculid mites and is endemic to East and Southeast Asia and Northern Australia. The clinical syndrome classically consists of a fever, rash, and eschar, but scrub typhus also commonly presents as an undifferentiated fever that requires laboratory confirmation of the diagnosis, usually by indirect fluorescent antibody (IFA) assay. We discuss the limitations of IFA, debate the value of other methods based on antigen detection and nucleic acid amplification, and outline recommendations for future study.Scrub typhus (Orientia tsutsugamushi infection) is transmitted by the bite of larval trombiculid mites and is endemic to the land mass within the triangle bounded by Japan to the north, Northern Australia to the south, and the Arabian Peninsula to the west. Mortality in the pre-antibiotic era was variable and in some series, approached 60%,¹ but specific and effective antimicrobial chemotherapy is now available.Scrub typhus often presents as fever with little to distinguish it clinically from co-endemic diseases such as typhoid, leptospirosis, and dengue. The presence of an eschar supports the diagnosis but is variably present.² Diagnosis, therefore, depends on clinical suspicion, prompting the clinician to request an appropriate laboratory investigation, and failure to diagnose the disease will likely result in treatment with ineffective β-lactam–based regimens.The mainstay in scrub-typhus diagnostics remains serology. The oldest test in current use is the Weil–Felix OX-K agglutination reaction, which is inexpensive, easy to perform, and results are available overnight; however, it lacks specificity and sensitivity³ (4 Indirect immunoperoxidase (IIP) eliminates the expense of a fluorescent microscope by substituting peroxidase for fluorescein (3

Table 1

Comparison of the accuracy and performance characteristics of assays for acute diagnosis of scrub-typhus infection

In vitro Screening of Compounds against Laboratory and Field Isolates of Human Hookworm Reveals Quantitative Differences in Anthelmintic Susceptibility

A panel of 80 compounds was screened for anthelmintic activity against a laboratory strain of Ancylostoma ceylanicum and field isolates of hookworm obtained from school children in the Kintampo North District of the Brong Ahafo Region of Ghana. Although the laboratory strain of A. ceylanicum was more susceptible to the compounds tested than the field isolates of hookworm, a twofold increase in compound concentration resulted in comparable egg hatch percent inhibition for select compounds. These data provide evidence that the efficacy of anthelmintic compounds may be species-dependent and that field and laboratory strains of hookworm differ in their sensitivities to the anthelmintics tested. These data also suggest that both compound concentration and hookworm species must be considered when screening to identify novel anthelmintic compounds.Human hookworm disease results from infection by two genera of hookworms, Ancylostoma spp. and Necator americanus. Albendazole (ABZ) and mebendazole (MBZ) are the primary drugs used to treat hookworm infection. A recent meta-analysis and review of field-based studies have shown that single-dose treatment cure rates with ABZ and MBZ are low (72% and 15%, respectively).^1,2 Compounding these treatment failure rates in humans is the well-documented emergence of drug resistance to ABZ and MBZ among parasitic nematodes of agricultural and veterinary importance.^3–5 In light of this information, the development of novel anthelmintic chemotherapeutics is necessary.Currently, the identification and development of novel anthelmintic compounds to cure hookworm infection rely on laboratory-based screening for inhibitors of egg hatching, larval motility and morphology, and/or adult worm survival.^6–9 However, novel compounds identified using small animal models of hookworm infection are rarely screened for comparable activity against field isolates of human hookworms.^10–16 Therefore, the correlation between the anthelmintic activity of compounds against laboratory and field isolates of human hookworms remains largely unknown.Moreover, for those cases in which anthelmintic activity is investigated, the larvicidal, rather than ovicidal, properties of a compound are usually analyzed.^17–20 Ovicidal activity is more frequently and most readily assayed in resource-limited field settings, where larval and adult hookworm-based assays are impractical tools for assessing anthelmintic activity.^21–23 In this study, we investigated the anthelmintic activity of a panel of compounds against laboratory and field isolates of hookworm using a standard egg hatch assay (EHA).^6,7 These data support the use of the EHA in field investigations for anthelmintic discovery and provide evidence for the continued development of the active compounds that we identified as novel anthelmintics.^22,24,25In total, 80 compounds were tested for anthelmintic activity against laboratory and field isolates of human hookworm using the EHA; 41 of the compounds were analogs of furoxan, an oxadiazole 2-oxide with proven nematicidal activity against the laboratory strain of A. ceylanicum.²⁶ An additional 14 compounds were analogs of furoxan that had not been previously tested against a laboratory strain of hookworm. The furoxan analogs were synthesized at the National Institutes of Health Chemical Genomics Center as part of the Therapeutics for Rare and Neglected Diseases Program.²⁷ To increase the diversity of chemotypes tested, a wide range of compounds (25 in total) was selected to be screened. Some of these molecules were chosen based on their putative targets. As an example, ABT-263 (navitoclax; Abbott Laboratories, Abbott Park, IL) is a molecule that acts on the B-cell lymphoma 2 (Bcl-2) protein, a regulator of apoptotic activity and an important anticancer target. It was recently shown that a similar molecule, ABT-737 (referred to here as National Chemical Genomics Center [NCGC] 00249278-01), binds the Bcl-2 protein in Schistosoma mansoni and may promote parasite death.²⁸ The remaining compounds in the set were Food and Drug Administration (FDA)-approved anticancer and antimalarial drugs selected for their potential anthelmintic properties.For screening against A. ceylanicum, pooled fecal samples were harvested from Golden Syrian hamsters after infection by oral gavage with L3 stage A. ceylanicum as previously described.^8,29,30 Hookworm eggs were purified from feces using a density floatation method, and the mean number of eggs per milliliter was calculated. For screening against field isolates of hookworm, duplicate fecal samples were collected from 142 Ghanaian school-aged children selected from five communities previously identified as having a high prevalence of hookworm infection.³¹ Each sample was examined for the presence of hookworm eggs using the Kato–Katz fecal smear technique, and hookworm eggs from positive samples were purified as described above and pooled.³² Purified eggs were pipetted into 96-well plates (100 eggs per well) containing water followed by the addition of compound dissolved in dimethyl sulfoxide (DMSO). Every compound was tested in duplicate at a final concentration of either 100 or 200 μM. EHAs were incubated for 24 hours at ambient temperature. Water and ABZ served as the negative and positive controls, respectively. The numbers of larvae and unhatched eggs were counted by light microscopy, and percent egg hatch inhibition values were calculated as Of 80 compounds assayed, 20 compounds inhibited the hatching of A. ceylanicum by > 90% at 100 μM (​and2).2). When tested against hookworm field isolates at the same concentration, only eight compounds inhibited hatching by > 50%, and no compound exhibited > 86% inhibition of egg hatching. All compounds that inhibited egg hatching of hookworm field isolates by > 50% were found to be > 90% effective at inhibiting A. ceylanicum egg hatching. Increasing the compound concentration to 200 μM resulted in four additional compounds achieving egg hatch inhibition values over 50% (Compound ID numberEgg hatch inhibition (%)Laboratory isolates^*Field isolatesNCGC00167928-01^†100.076.4NCGC00167944-01^†100.067.2NCGC00167942-01100.039.0NCGC00167920-01100.016.9NCGC00167922-0199.425.8NCGC00182103-0199.21.8NCGC00167919-0199.219.1NCGC00241748-0198.90NCGC00183324-0198.821.8NCGC00167932-0197.97.4NCGC00182106-01^†97.958.0NCGC00167927-01^†97.858.2NCGC00167935-01^†97.172.2NCGC00168364-0196.12.2NCGC00167918-0195.946.1NCGC00167924-0195.010.3NCGC00242405-0192.73.7NCGC00168368-0192.09.3NCGC00094237-01^†90.256.1Open in a separate windowData shown for analogs exhibiting inhibition values above 90%.^*Percent values represent the mean of two independent screenings—one conducted before and one conducted after the field screening.^†Denotes all tested compounds that inhibited hatching of field isolates by more than 50%.

Table 2

Highest in vitro egg hatch inhibition values among NCGC compounds tested against the laboratory strain of A. ceylanicum and field isolates of human hookworms

Blood-Borne Candidatus Borrelia algerica in a Patient with Prolonged Fever in Oran,Algeria

To improve the knowledge base of Borrelia in north Africa, we tested 257 blood samples collected from febrile patients in Oran, Algeria, between January and December 2012 for Borrelia species using flagellin gene polymerase chain reaction sequencing. A sequence indicative of a new Borrelia sp. named Candidatus Borrelia algerica was detected in one blood sample. Further multispacer sequence typing indicated this Borrelia sp. had 97% similarity with Borrelia crocidurae, Borrelia duttonii, and Borrelia recurrentis. In silico comparison of Candidatus B. algerica spacer sequences with those of Borrelia hispanica and Borrelia garinii revealed 94% and 89% similarity, respectively. Candidatus B. algerica is a new relapsing fever Borrelia sp. detected in Oran. Further studies may help predict its epidemiological importance.Relapsing fever borreliae are arthropod-borne pathogens causing mild to deadly septicemia and miscarriage.¹ In Africa, cultured representatives include tick-borne Borrelia crocidurae, Borrelia duttonii, and Borrelia hispanica transmitted by Ornithodoros soft ticks and louse-borne Borrelia recurrentis.¹ Borreliae are fastidious bacteria responsible for various febrile presentations, most commonly malaria-like symptoms.^1,2 Borreliae have been documented in patients with tick-borne relapsing fever, however little is known regarding Borrelia in north Africa.² Borrelia crocidurae has been detected with a 2.5% prevalence in Ornithodoros sonrai ticks,³ while Lyme group Borrelia garinii was recently detected in Ixodes ricinus ticks, collected from El Ghora, Algeria.⁴ In addition, at least 10 different relapsing fever–causing borreliae have been documented in Africa, including five different borreliae in humans and five different borreliae in nonhuman hosts.² The former includes pathogens classified as B. hispanica, B. crocidurae, B. duttonii, and B. recurrentis.² Although relapsing fever–causing Borrelia may form one genetic species, they differ in their vector, host range, and disease spectra protein profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.^5–7 Accordingly, molecular tools can be used to discriminate these different Borrelia.^8,9 Here, using such molecular tools, we detected sequences indicative of a new Borrelia sp. named Candidatus Borrelia algerica in a blood sample from a patient with prolonged fever in Oran, Algeria.We studied 257 blood samples collected from febrile patients in Oran between January and December 2012. Interviews, sampling (3–4 mL blood in ethylenediaminetetraacetic acid [EDTA] tubes) and a medical examination were performed on each individual with a fever (an axillary temperature > 37.5°C) and a questionnaire was completed by each patient. We have previously reported the presence of Coxiella burnetii,¹⁰ Rickettsia felis, and Plasmodium spp. in this patient series.¹¹ A 200 μL sample of whole blood was used for DNA extraction performed using a QIAamp DNA Micro Kit according to the manufacturer protocols (Qiagen, Hilden, Germany). The samples were handled appropriately to avoid cross-contamination. The quality of the DNA handling and extraction was verified by real-time polymerase chain reaction (RT-PCR) for the housekeeping gene encoding beta-actin¹² (8^,9 Two sets of negative controls (DNA of blood from a nonfebrile patient and sterile water) and a positive control (B. recurrentis DNA) were also analyzed in each run. All positive and negative controls demonstrated the expected results in all tests and Borrelia spp. were detected in four (1.6%) patients.

Table 1

Primers and probes used in this study

Infection Dynamics of Sylvatic Dengue Virus in a Natural Primate Host,the African Green Monkey

The four serotypes of mosquito-borne dengue virus (DENV-1, -2, -3, and -4) that circulate in humans each emerged from an enzootic, sylvatic cycle in non-human primates. Herein, we present the first study of sylvatic DENV infection dynamics in a primate. Three African green monkeys were inoculated with 10⁵ plaque-forming units (pfu) DENV-2 strain PM33974 from the sylvatic cycle, and one African green monkey was inoculated with 10⁵ pfu DENV-2 strain New Guinea C from the human cycle. All four monkeys seroconverted (more than fourfold rise in 80% plaque reduction neutralization titer [PRNT₈₀]) against the strain of DENV with which they were inoculated; only one (33%) of three monkeys infected with sylvatic DENV showed a neutralizing antibody response against human-endemic DENV. Virus was detected in two of three monkeys inoculated with sylvatic DENV at low titer (≤ 1.3 log₁₀pfu/mL) and brief duration (≤ 2 days). Clinical signs included rash and elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels.Mosquito-borne dengue virus (DENV; genus Flavivirus) is one of only two arthropod-borne viruses to have established a transmission cycle endemic to humans that is ecologically and evolutionarily distinct from its enzootic ancestors.¹ In its human transmission cycle, the virus comprises four antigenically and genetically distinct serotypes (DENV-1, -2, -3, and -4); infection with one serotype conveys lifelong protection against homologous challenge but only transient protection against heterologous infection with another serotype.^2,3 Although most infections are subclinical, a fraction results in classical dengue fever (DF), a self-limited febrile illness, and some of these patients progress to severe dengue disease.⁴ The lack of an animal model that recapitulates human dengue disease has been a major barrier to the development of DENV vaccines and therapeutics. Replication of human-endemic DENV in non-human primates (NHPs)⁵ is muted in intensity and duration relative to replication in humans,⁶ and infection with human-endemic DENV produces disease in NHPs only when administered at doses that greatly exceed those delivered by the mosquito.¹Each of four human-endemic DENV serotypes emerged from enzootic ancestors maintained in a sylvatic cycle between NHPs and canopy-dwelling Aedes mosquitoes.^1,7 These sylvatic cycles remain active in the forests of southeast Asia and west Africa. Spillover of sylvatic DENV into humans, sometimes causing severe disease, has been repeatedly documented.^8–15 Thus, continued circulation of sylvatic DENV may threaten future control of human DENV when a DENV vaccine becomes available.⁷ Because the replication and immunogenicity of sylvatic DENV in its NHP hosts have not previously been investigated, some mathematical models of sylvatic DENV population dynamics and spillover risk have used infection parameters derived from studies of human-endemic DENV in NHPs.¹⁶ However, infection dynamics of arboviruses in reservoir and non-reservoir hosts can differ substantially.^17,18 Yellow fever virus (YFV) offers a particularly dramatic example of this difference: YFV infection rarely causes overt illness in African NHPs that serve as reservoir hosts of its ancestral sylvatic cycle, whereas YFV infection of New World monkeys results in high rates of disease and death (reviewed in ref. 1). Mandl and others¹⁹ showed that the YFV-17D live-attenuated vaccine strain replicated to lower levels and for shorter duration in YFV reservoir (sooty mangabeys [Cercocebus atys]) than novel (rhesus macaques [Macaca mulatta]) hosts. Moreover, neutralizing antibody responses to YFV-17D were significantly lower in sooty mangabeys than in rhesus macaques 28 days post-infection (pi); neutralizing antibodies waned to undetectable levels by 120 days pi in sooty mangabeys but were maintained at high levels in rhesus macaques. If replication of sylvatic and human-endemic DENV in NHPs differs to a similar degree, models of sylvatic DENV transmission dynamics that rely on infection dynamics of human-endemic DENV in NHPs may be misleading.In this study, we infected African green monkeys (AGMs; Chlorocebus sabaeus), a known host of sylvatic DENV-2 in West Africa,¹ with a West African sylvatic strain of DENV-2. AGMs were provided by Primate Products (Miami, FL) from a colony maintained in St. Kitts. Sixteen adults were first screened by plaque reduction neutralization titer (PRNT) essentially as previously described^20,21 to ensure that they had not been exposed to DENV-1, -2, -3, or -4, YFV, or West Nile virus. All 16 NHPs were seronegative (PRNT₈₀ ≤ 20) for all viruses. Four males weighing > 6 kg were then quarantined in the Tulane National Primate Research Center (TNPRC) in Covington, Louisiana. All procedures using these animals were performed by the clinical veterinary staff at the TNPRC under the guidance of veterinarians, and they were approved by the Institutional Animal Care and Use Committee (IACUC) of Tulane University in compliance with the American Association for Laboratory Animal Science (AALAS) “Policy on the Human Care and Use of Laboratory Animals.” Monkeys were anesthetized with ketamine at an intramuscular (i.m.) dose of 10 mg/kg body weight on day 0 of the study; three AGMs were inoculated i.m. with 10⁵ plaque-forming units (pfu) sylvatic DENV-2 strain PM33974, and one AGM was inoculated i.m. with 10⁵ pfu DENV-2 strain New Guinea C (NGC) from the human cycle. The dose was chosen to enable comparisons with previous studies of human DENV²² and other flaviviruses¹⁹ in NHPs and DENV vaccine candidates in humans.²³ Inocula were administered in a total volume of 1 mL, with 0.5 mL administered to each upper arm.Sylvatic DENV-2 strain PM33974 was isolated from a pool of Ae. africanus mosquitoes in Guinea in 1981 by inoculation into Toxorhynchites amboinensis mosquitoes and subsequently passaged five times in Ae. albopictus C6/36 cells to create the stock used for infections. This strain has been studied extensively in cell culture and mouse models of human infection.^24,25 DENV-2 NGC was derived from the prototype strain (isolated in 1944) without passage in mouse brains.²⁶ DENV-2 NGC was chosen as a control, because in a previous experiment, this strain produced viremia in 100% of rhesus macaques (M. mulatta) injected with 10⁵ pfu.²² The complete genome sequence of the DENV-2 PM33974 lineage used in this study has been determined (Genbank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF105378.1","term_id":"132271144","term_text":"EF105378.1"}}EF105378.1),²⁴ but only the structural genes (capsid, pre-membrane, and envelope genes; 807 amino acids in total) of the DENV-2 NGC lineage used in this study have been sequenced ({"type":"entrez-nucleotide","attrs":{"text":"AY243468.1","term_id":"30026603","term_text":"AY243468.1"}}AY243468.1).²² The structural genes of the two strains showed 93% amino acid identity. To compare whole genomes, the sequence of a different strain of DENV-2 NGC ({"type":"entrez-nucleotide","attrs":{"text":"AF038403","term_id":"2723944","term_text":"AF038403"}}AF038403) was aligned to the complete genome of DENV-2 PM33974. The two sequences showed 82% nucleotide identity across the entire genome, 94% amino acid identity in the coding region, and 92% nucleotide identity at both the 5′ and 3′ untranslated regions. To compare the replication of the two viruses in cell culture, plaque size of each virus was measured in C6/36 cells (9 plaques/virus) and AGM kidney Vero cells (30 plaques/virus) as previously described.²⁷ DENV-2 NGC produced substantially smaller plaques than DENV-2 PM33974 in C6/36 cells (mean in millimeters ± 1 SE = 0.29 ± 0.001 versus 0.40 ± 0.001; Student''s t test, degree of freedom [df] = 16, P < 0.0001) but slightly larger plaques than DENV-2 PM33974 in Vero cells (0.19 ± 0.002 versus 0.15 ± 0.012; Student''s t test, df = 58, P = 0.045).Serum was collected on days 1–10, 12, 14, 16, and 18 pi to monitor viremia and days −3 and 28 pi to assay neutralizing antibodies by PRNT₈₀ against DENV-2 strains NGC and PM33974. Blood was drawn to conduct a complete blood count and measure components of serum biochemistry (bilirubin, alkaline phosphatase, creatinine, creatinine phosphokinase, glucose, aspartate aminotransferase [AST], alanine aminotransferase [ALT], blood urea nitrogen [BUN], and albumin/globulin ratio) and electrolytes (Na, Cl, and K) on study days −3, 4, 7, and 28 pi. Rectal temperature, behavior, vital signs, weight, and skin condition were monitored on days −3, 0–10, 12, 14, 16, 18, and 28 pi. Viremia was quantified by serial dilution of serum and immunostaining in Vero cells as previously described^20,21; virus titers are shown in MonkeyDENV-2 transmission cycleDENV-2 strainViremia^* (titer [log₁₀pfu/mL]) and detection of rash on a specific day1234567891012141618JT08HumanNGC^††1.02.0^‡‡JT09SylvaticPM339741.31.3^‡‡JT10SylvaticPM33974^‡‡JT11SylvaticPM33974^†‡‡‡Open in a separate windowVirus was not detected by either method unless noted.^*Numbers indicate virus titer from direct assay of serum.^†Detection of virus after one passage of serum in Vero cells.^‡Rash detected.All four monkeys seroconverted (more than fourfold rise in PRNT₈₀) with high PRNT₈₀ titers against the strain of DENV with which they were inoculated (28 However, of the three monkeys infected with sylvatic DENV-2, only one (33%) monkey seroconverted to DENV-2 NGC. Blaney and others²⁹ have also reported variation in neutralization of different strains of DENV within a serotype. Blaney and others²⁹ tested the neutralizing activity of serum from vaccinees who received a live-attenuated tetravalent dengue vaccine against five different DENV strains per serotype, including each of the four strains incorporated into the vaccine. The study found, for each serotype, that (1) the vaccine (infecting) strain was robustly neutralized, (2) at least one strain was more efficiently neutralized than the vaccine strain, and (3) although all strains of DENV-1, -2, and -4 were efficiently neutralized, two of five DENV-3 strains were not.²⁹ The study by Blaney and others²⁹ and other studies^30,31 have raised concerns that there could be gaps in vaccine protection caused by strain variation. Similarly, the asymmetry in neutralization of DENV-2 NGC by monkeys infected with DENV-2 PM33971 suggests that NHP populations could be susceptible to some strains of human DENV, even if previously exposed to the homologous serotype of sylvatic DENV. DENV has been shown to spill back from humans into NHPs,³² and such spillback could result in the formation of new sylvatic cycles.¹

Table 2

Neutralizing antibody responses of AGMs 3 days pre-infection and 28 days p.i. with designated strains of DENV-2

The CyScope® fluorescence microscope, a reliable tool for tuberculosis diagnosis in resource-limited settings

Poor laboratory equipment and few human resources have made it difficult to implement microscopic diagnosis of pulmonary tuberculosis (TB) on a large scale basis worldwide. Three hundred sputum samples from patients in Cameroon were studied by using the CyScope^®, a new light-emitting, diode-based, fluorescence microscope, to compare auramine-rhodamine fluorescence with the conventional Ziehl-Neelsen staining method. Five fluorescence protocols were tested to reduce manipulation time. Smear positivity for acid-fast bacilli with the Ziehl-Neelsen staining method was 27.7% (83 of 300) compared with 33.3% (100 of 300) with the fluorescent method. Staining time with the modified fluorescence protocol could be reduced from 21 minutes to 10 minutes. This study confirmed that the fluorescence staining method is more sensitive than the Ziehl-Neelsen staining method. It is suggested that the training of laboratory technicians on fluorescence microscopy should be scaled up for increased disease control.In the context of human immunodeficiency virus/acquired immunodeficiency syndrome pandemics, tuberculosis (TB) is the most common opportunistic infection. More than 9 million cases in 2007 were found in Africa and Southeast Asia.¹ The absence of accurate diagnostic techniques constitutes a serious hindrance in the strategy of TB control in Africa.² The increase of false negative-smear pulmonary TB is highlighted as one of the main reasons for the upsurge of this disease during the past decade in poor countries.³In an effort to improve methods to diagnose pulmonary tuberculosis, the World Health Organization has encouraged the development of simplified and accurate diagnostics, such as fluorescence microscopy for early detection of Mycobacterium tuberculosis in sputum.⁴ The CyScope^® (Partec, Görlitz, Germany) belongs to a new generation of fluorescence microscopes that use light-emitting diodes (LEDs) as a light source.⁵ It can be plugged into an ordinary electrical outlet or operated with a built-in rechargeable battery. It is equipped with a high power Royal Blue LED (wavelength = 455 nm) for incidence fluorescence excitation and a white light LED for transmitted light. The LED fluorescence microscope provided similar results as standard mercury vapor lamp fluorescence microscope at a much lower cost.⁶ In this study, we used the LED fluorescence microscope CyScope^® for TB diagnosis and investigated different staining procedures to shorten the handling time for samples.During August–November 2009, 300 sputum samples were collected from patients in Cameroon with suspected pulmonary TB or patients receiving treatment. The age range of the patients was 2–74 years. Two slides were prepared from the same specimen for direct sputum smears by using the unconcentrated specimens in the solid or most dense particles of the sputum.⁷ The smears were dried in air and heat-fixed. Staining was classically processed as described by the World Health Organization and the International Union Against Tuberculosis and Lung Disease. A solution with 0.3% basic fuchsin (pararosaniline chloride with 88% P1528 dye; Sigma, St. Louis, MO) was heated to the steaming point, decolorized with 25% sulfuric acid, and counterstained with 0.3% methylene blue.^7,8The fluorescent method of Degommier⁹ was applied. The staining reagent Tb-fluor (Merck, Darmstadt, Germany) was used according to the manufacturer''s procedure. Briefly, smears were stained with auramine-rhodamine, rinsed with tap water, decolorized with HCl-isopropanol, and counterstained with KMnO₄. Five protocols with variable staining and counterstaining durations were used to establish overall minimal staining time with the fluorescent dye. (ProtocolsSteps (minutes)Duration (minutes)Auramine-rhodamineHydrochloric acid and isopropanolPotassium permanganateI151521II101516III101112IV81110V4116Open in a separate windowAcid-fast–stained smears were colored by a variety of protocols and observed on the CyScope^®. This microscope uses white light and fluorescence. When the fluorescence function was turned on, the acid-fast bacilli were visualized at magnifications of 200× and 400×. Their number in the expectoration fluid was related to the sample contagiousness, and the result was expressed quantitatively.^1,7Solid Löwenstein-Jensen medium containing pyruvate was considered as the gold standard^4,10,11 for samples with discrepant microscopic results. Each sputum sample was liquefied and decontaminated with 3% sodium laurylsulfate and 1% NaOH and neutralized with H₂SO₄-bromocresol purple solution.¹² Two slants of Löwenstein-Jensen medium were simultaneously inoculated with each specimen and incubated at 37°C for 8 weeks. Identification of TB strains was based on growth rate and colony morphology.¹³Data were analyzed by using the database/statistical package SPSS version 10.0 (SPSS Inc., Chicago, IL). The chi-square test and proportion test for a binomial distribution were used to compare differences between the fluorescent method and the Ziehl-Neelsen method. Statistical analysis was conducted with a 5% significance level.A total of 300 sputum specimens were included in the study. Of these specimens, 17 (6%) of Ziehl-Neelsen–negative samples were positive under by the fluorescent method and confirmed by Löwenstein-Jensen culture (11^,14–17

Table 2

Comparison of the Ziehl-Neelsen method with the fluorescent method for the detection of acid-fast bacilli

Mycobacterium tuberculosis Isolates from Single Outpatient Clinic in Panama City Exhibit Wide Genetic Diversity

Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities.Tuberculosis (TB) affects nearly 8.7 million people worldwide.¹ In 2011, most TB cases were reported in Asia (59%) and Africa (26%), although cases were reported to a lesser extent in the Eastern Mediterranean Region (7.7%), the European Region (4.3%), and the Americas Region (3%). Panama stands as the country with the highest TB mortality rate in Central America.² In 2012, more than 1,500 TB cases were reported in Panama, for an average incidence rate of 43.1 cases per 100,000 inhabitants.³ Areas located at the Pacific and Atlantic entries of the Panama Canal have harbored the highest numbers of TB cases since the Canal''s construction.⁴ Despite sanitation improvements in terminal port cities, recent studies have revealed elevated TB transmission as a result of a high clustering rate among multidrug-resistant TB cases.^5,6 However, data on the transmission of drug-susceptible TB within the general population remain scarce and have not been updated to reflect a second wave of immigration connected with Panama Canal expansion activities.⁷Mycobacterium tuberculosis genotyping has proven to be the most important laboratory tool in understanding TB transmission.⁸ In addition to studies on patient contacts; information on molecular epidemiology is useful for evaluating TB control program results. Genotyping also assists in monitoring molecular markers associated with virulence, immunogenicity, and drug resistance⁹; among the genotyping tools available, the IS6110-restriction fragment length polymorphism (RFLP) reference standard method is based on the number of repetitions of the IS6110 sequence along the M. tuberculosis genome.¹⁰ This tool discriminates between clonally related and unrelated isolates. On the other hand, Spoligotyping focuses on detecting 43 spacer sequences in the direct repeat region of the M. tuberculosis genome. Unfortunately, the IS6110–RFLP method is a complex and laborious procedure, whereas Spoligotyping is faster and simpler but less discriminating.^11,12 The study of mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) is an alternative to genotyping M. tuberculosis isolates.^13,14 Our study aimed to characterize the genetic diversity of M. tuberculosis isolates in one outpatient clinic using a combination of 12 loci MIRU-VNTR.A total of 62 clinical isolates were collected at the Social Security Clinical Laboratory of the Complejo Hospitalario Metropolitano Dr. Arnulfo Arias Madrid between January and December of 2005. The strain collection was performed as part of the Panamanian standard of patient care for TB diagnosis and control in Panama City. These isolates accounted for 16.3% of all pulmonary TB cases reported in Panama City in 2005. The DNA extraction was performed using a method described previously.¹⁰ A total of 12 MIRU-VNTR loci were amplified according to a modified protocol described by Cowan and colleagues.¹³ The amplification products were analyzed by electrophoresis on an agarose gel. The number of MIRU-VNTR alleles was determined according to the sizes proposed by Cowan and colleagues, which allocate the number of alleles by the fragment size.¹³ The allelic diversity for each MIRU-VNTR was calculated using the number of alleles at each locus.¹⁵ The ability to detect the number of allelic repetitions of each allele for every MIRU-VNTR was then classified as high, moderate, or low. We used the Hunter-Gaston discriminatory index (HGDI) to determine the discriminating power of possessing all 12 MIRU -VNTR loci in our study.¹⁶ Spoligotyping was performed on genomic DNA using the standard method described by Kamerbeek and colleagues.¹⁷ The family label and Spoligotype octal code numbers were obtained from the SPOLDB4.0.¹⁸Our findings show high genetic diversity of M. tuberculosis clinical isolates obtained from outpatients from a clinic in Panama City. Our results reveal that a total of 45 (72.5%) M. tuberculosis isolates showed unique MIRU-VNTR patterns (19 These findings indicate the genetic diversity of M. tuberculosis circulating in patients with drug-susceptible pulmonary TB in an outpatient clinic in Panama City. Further detailed studies are needed to determine the connection between patients with isolates in the same clusters.

Table 1

MIRU-VNTR genotypes for M. tuberculosis clinical isolates with a unique genotype recovered from an outpatient clinic in Panama City (2005)^*

Genetic Variation of Echinococcus canadensis (G7) in Mexico

We evaluated the genetic variation of Echinococcus G7 strain in larval and adult stages using a fragment of the mitochondrial cox1 gen. Viscera of pigs, bovines, and sheep and fecal samples of dogs were inspected for cystic and canine echinococcosis, respectively; only pigs had hydatid cysts. Bayesian inferences grouped the sequences in an E. canadensis G7 cluster, suggesting that, in Mexico, this strain might be mainly present. Additionally, the population genetic and network analysis showed that E. canadensis in Mexico is very diverse and has probably been introduced several times from different sources. Finally, a scarce genetic differentiation between G6 (camel strain) and G7 (pig strain) populations was identified.Echinococcus granulosus sensu lato (s.l.) includes species that cause cystic echinococcosis (CE), one of the most important and widespread parasitic zoonoses. Recent phylogenetic studies based on both mitochondrial and nuclear DNA genes show that E. granulosus s.l. consists of at least four valid species: E. granulosus sensu stricto (s.s.; genotypes G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). Genotypes G6/G7 are closely related and referred to as camel and pig strains, respectively.^1–3 The pig–dog cycle is mainly present in Mexico and maintains the G7 strain.^4,5 Although there are isolated reports of E. oligarthrus in a wild cat,⁶ E. ortleppi (E. granulosus s.l.; G5) in a patient,⁷ and E. granulosus s.s. (G1) in a rural pig, there is no evidence that these species are maintained in Mexico.⁸ No data of CE caused by G7 have been documented in Mexican patients, although there is a high number of E. canadensis G7-infected patients in central Europe, pointing to the importance of this strain as a cause of human CE.^9,10 There are only two genetic studies performed in samples from Mexico. Cruz-Reyes and others⁵ documented that G7 parasites of Mexican and Polish pig isolates showed similar patterns by restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) and random amplified polymorphic DNA (RAPD) techniques, and although polymerase chain reaction (PCR) -sequencing analysis of mitochondrial cox1 gen fragment was performed, no polymorphism data were reported. Sharma and others¹¹ identified two variants (A and B) inside of the G6/G7 group consisting of samples from Mexico and Argentina using five nuclear markers (elongation factor 1α, transforming growth factor-β receptor kinase, thioredoxin peroxidase, calreticulin, and ezrin-radixin-moesin-like protein). Because some local slaughter records from northern Mexico indicate the presence of Echinococcus spp. in livestock animals,⁵ the objective of this study was to investigate if parasites in pigs and dogs correspond to G7 and if so, describe its genetic variation.Infected animals were identified in the municipal slaughterhouse of Calera, Zacatecas (north central Mexico), where farm and backyard livestock animals coming from the whole state and other surrounding states were included. For this purpose, viscera from 387 pigs, 243 bovines, and 32 sheep were inspected for the larval stage of Echinococcus. Nine pigs (six pigs from Zacatecas, two pigs from Aguascalientes, and one pig from Morelos) were found infected, and hydatid cysts were obtained under aseptic conditions. After cyst contents were aspirated and centrifuged, aliquots were examined under microscopy to confirm the presence of protoscolices, and pellets were kept in 70% ethanol at −20°C until DNA extraction. Each cyst from each animal was considered as an isolate.Based on the presence of the parasites previously identified in Calera''s slaughterhouse, a rural community located in the central area of Zacatecas at 22°55′ N, 102°48′ W was selected to look for the adult stage of this parasite. For this search, all dogs (60) present in the community were sampled one time for feces after obtaining verbal consent from the owner; samples were used to identify taeniid eggs by the Faust technique, antigens in stool samples (copro-antigens) by enzyme-linked immunosorbent assay (ELISA; CpAg ELISA), and DNA by Copro-PCR. The CpAg ELISA was performed as described by Allan and others¹² and Moro and others.¹³ For Copro-PCR, only positive samples by CpAg ELISA were analyzed using JB3 and JB4 primers to amplify a cox1 gen fragment.¹⁴ Coprological analysis of dogs showed that 11 samples were positive by CpAg ELISA (18.3%); only 2 of these samples had taeniid tapeworms (3.4%), and 3 of 11 samples yielded products of approximately 450 bp. All amplicons obtained of hydatid cysts and fecal samples were purified, sequenced on both strands, submitted to GenBank (accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"KF734649-KF734660","start_term":"KF734649","end_term":"KF734660","start_term_id":"570963772","end_term_id":"570963794"}}KF734649-KF734660), and compared with several mitochondrial DNA sequences of cox1. Dogs positive for taeniid eggs or antigens were purged and treated with praziquantel at 30 mg/kg and arecoline bromide at 2 mg/kg. The protocol was previously approved by the Ethics and Research Committees of the General Hospital “Dr. Manuel Gea Gonzalez”; government and health authorities of the municipality and community also authorized our study.All sequences were subjected to the Basic Local Alignment Search Tool (BLAST) search in the GenBank database; multiple alignments were performed with the CLUSTAL W and MUSCLE programs,^15,16 with manual adjusted in MEGA program v5¹⁷ to determine the appropriate model of molecular evolution in the Modeltest 3.7 program.¹⁸ The phylogenetic reconstruction using Bayesian inference was performed with Mr Bayes 3.2.1 program.¹⁹ Unrooted haplotype networks were created using NETWORK 4.611 software and nested according to the rules in median-joining networks.²⁰ An analysis of genetic diversity within and between populations was performed using DnaSPv4²¹ and included nucleotide diversity (π), haplotype polymorphism (θ), genetic differentiation index (F_ST), and Tajima''s D test. Analysis of molecular variance (AMOVA) was used to examine the population genetic structure between populations by Φ_ST as the genetic fixation index (analogous to F_ST) obtained by ARLEQUIN software.²²After multiple alignments, all sequences of larval and adult stages showed 98% or higher identity with E. canadensis, whereas the Bayesian phylogenetic tree and the haplotype network inference grouped these sequences in the E. canadensis G7 cluster. Sequences for cox1 of E. canadensis from Africa, Asia, Europe, Latin America, and North America deposited in the GenBank databases (N = 58) as well as our sequences (accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"KF734649-KF734660","start_term":"KF734649","end_term":"KF734660","start_term_id":"570963772","end_term_id":"570963794"}}KF734649-KF734660) were analyzed. The results for π and θ were 0.0118 and 0.718, and the result of Tajima''s D test was −2.1885 (P < 0.01). Genetic differentiation indexes between different paired sequences of E. canadensis genotypes are shown in Population APopulation BF_STAMOVAReferencesΦ_STSSVCPercentG6G70.0310.0851.6400.0608.530–³⁸G6G80.8930.93737.7675.39593.7G6G100.6240.61315.7980.72661.3G7G80.7830.76027.2504.31576.030,31,39,40G7G100.3590.3368.7220.53233.6G8G100.8820.88140.0255.99188.130,34,36,39Mexico (G7)Europe (G7)0.2010.1793.4940.25917.930,31,40,41Latin America (G7)Europe (G7)0.1460.1132.4610.13811.3Latin America (G7)Africa (G6)0.1470.1543.3340.17115.431,33,35Latin America (G7)Asia (G6)0.1560.1262.7220.14412.630,31Latin America (G7)Africa–Asia (G6)0.1510.2053.8330.18020.630,31,33,35Europe (G7)Africa (G6)0.0470.0430.7270.0224.330,33,35,40,41Europe (G7)Asia (G6)0.0610.0190.4720.0249.130,40,41Europe (G7)Africa-Asia (G6)0.0420.0600.6500.2336.030,33,35,40,41Open in a separate windowEurope (G7) includes G7 sequences from Italy, Poland, and Romania. Latin America (G7) includes G7 sequences from Mexico and Peru. Africa (G6) includes G6 sequences from Algeria, Ethiopia, Mauritania, and Sudan. Asia (G6) includes G6 sequences from Iran and Kazakhstan. Africa–Asia (G6) includes G6 sequences from China, Iran, Mauritania, Mongolia, and Russia. SS = sum of squared; VC = variance of components.For the network analysis, haplotypes of E. canadensis (G6, G7, G8, and G10), according to their hosts and country of origin, were included and exhibited three relevant dispersion centers (clustering more than nine haplotypes in each one of them): one for G10 from North America with elk/wolf, one for G6/G7 from Iran, Mauritania, and Peru with camel and sheep, and one for G6/G7 from Africa, Asia, and Latin America with cattle, camel, dog, elk, goat, and human. Interestingly, some G7 pig haplotypes from Mexico are displayed around the third dispersion center; in contrast, other G7 haplotypes from European and Asian countries are clustered around the second dispersion center (Figure 1).Open in a separate windowFigure 1.Haplotype network for E. canadensis using cox1 sequences of different countries and hosts. Numbers on branches refer to mutational changes. Sizes of circles are proportional to haplotype frequencies (numbers of haplotypes are shown inside circles). Thus, major circles represent ancestral haplotypes, and small circles represent missing haplotypes. Hosts are shown on a side of the haplotypes, and the three big ellipses with discontinuous lines containing G6/G7, G8, and G10.The sequences obtained from three dogs and nine infected pigs showed that E. canadensis (G7) was the only strain identified, indicating that it is the main genotype present in Mexico, which had been previously reported.^4,5 This study also shows that E. canadensis (G6, G7, G8, and G10) is lightly more polymorphic than other species of the genus Echinococcus (π = 0.0118), and the negative value of Tajima''s D test suggests a recent expansion for the populations. Haag and others²³ reported π = 0.0005 for E. multilocularis and π = 0.0090 for E. granulosus using mitochondrial (nad) and nuclear (ActII, Hbx2, and AgB) sequences; in addition, Sharma and others¹¹ performed a population genetic analysis of E. granulosus s.s. using cox1 sequences and found that π ranged from 0.0039 to 0.0093 for E. granulosus s.s. isolates from India, and they also found a negative value for Tajima''s D test. Small sample sizes and lengths of the nucleotide sequences might affect the π values, showing a tendency toward underestimation.²⁴ In addition, most studies of genetic variation in Echinococcus have used around a dozen sequences; therefore, π results might not be directly comparable among them. However, even under these considerations, this comparison allows us to highlight the genetic diversity among populations of E. canadensis. Furthermore, we found that, in E. canadensis populations, G6 and G7 have a scarce differentiation (F_ST and Φ_ST close to 0.1), whereas it is high for E. canadensis G8 and G10 (F_ST and Φ_ST > 0.6). In contrast, in a study focused on the genetic diversity of E. granulosus s.s., hydatid cysts from four European countries (Bulgaria, Hungary, Romania, and Italy) were evaluated by sequences of cox1 and showed F_ST values up 0.187.²⁵ In this study, when G6 and G7 were divided in geographic areas, a similar genetic differentiation was observed with F_ST and Φ_ST < 0.1, except when Latin America (G7) was matched with Europe, Africa, or Asia (F_ST and Φ_ST = 0.15–0.2), suggesting that the former population reflects a great genetic differentiation regarding the latter populations. This is strengthened by the network analysis, in which some haplotypes of pigs from Mexico are clustered in different branches from those from pigs of European countries.Based on the network analysis, we might deduce the following inferences. (1) E. canadensis G7 in Mexico is very diverse and has probably been introduced from abroad several times from different sources (i.e., Figure 1 shows that six Mexican isolates have from 4 to 14 mutational changes between the isolate and the main haplotype). (2) Haplotypes grouped in the North American wildlife cluster (G10) are closer within them (with one or two mutational changes), and they are placed far away from Mexican isolates; thus, they might be ruled out as sources of introduction to Mexico. (3) Differentiation between G6 and G7 would not make any sense based on the differentiation of genetic indexes found for both genotypes (F_ST and Φ_ST close to 0.1). Additionally, one of the main ancestral dispersion centers in the network analysis clustered identical haplotypes of G6 and G7 from China, Mexico, Peru, Sudan, and Russia. The species status of E. canadensis is still controversial,^1–3,5,25 because biologically different strains (G6–G10) have been unified. The camel (G6) and pig (G7) strains (both maintained primarily by dog-mediated domestic lifecycles from tropical to temperate zones) are ecologically and geographically segregated from G8 to G10^2,26; therefore, some works have suggested that G6 and G7 should be treated as a single species: E. intermedius.^5,27 However, in recent taxonomic revisions, this proposal has been considered inappropriate,^2,26 and the specific name of E. canadensis seems to be the most suitable for handling the closely related genotypes. Thompson and Lymbety²⁸ have argued that knowledge of the genetic structure of cestodes can be applied to the epidemiology and the control of these parasites, because genetic variation within and between populations determines future evolutionary changes, genetic differentiation, and speciation. According to our results, it is probable that E. canadensis G7 has been accidentally introduced from abroad several times through different sources, except from North America (where G10 is more prevalent). This knowledge may have important implications for control of the zoonosis, mainly in areas that lack adequate veterinary control, which could prompt an important health problem. Although presently there are few cases of human cystic echinococcosis in Mexico, interestingly, a study performed in a rural community where an autochthonous human case of CE was detected in 2006 showed that, although some risk practices (such as feeding dogs with infected viscera) were observed, no data of CE in livestock and canine echinococcosis were found, suggesting that CE in Mexico has an unclear pattern.²⁹     

Multiple Zoonotic Pathogens Identified in Canine Feces Collected from a Remote Canadian Indigenous Community

Five genera of potentially zoonotic bacteria and parasites were detected in environmentally collected fecal samples from a remote indigenous community in Northern Saskatchewan, Canada. Organisms identified include Toxocara canis, Echniococcus granulosus, Giardia duodenalis, Cryptosporidium spp., and Campylobacter spp. The prevalence and intensity of Giardia spp. and Campylobacter spp. in fecal samples was particularly remarkable. Three-quarters of samples tested contained at least one zoonotic species of Campylobacter, and C. jejuni-containing feces had an average of 2.9 × 10⁵ organisms/g. Over one-half of samples tested contained Giardia spp. with an average of 9,266 cysts/g. Zoonotic G. duodenalis Assemblage A was the only Giardia spp. genotype identified. These data suggest that canine feces have the potential to pose a significant health risk to Canadians in rural and remote indigenous communities.Domestic dogs have long been recognized to be a potential source of zoonoses for people.^1–4 In particular, zoonotic bacteria and parasites harbored in the canine intestine have been shown to pose a significant risk to human health.^1–4 People are exposed to these pathogens through direct or indirect contact with infected dogs or their feces, and they may become infected after inadvertent ingestion of a zoonotic agent.^2–4In Canada, indigenous people living in rural and remote communities seem to have an increased risk of exposure to and infection with certain canine fecal zoonoses compared with other Canadians.^5–7 This may be related to the fact that many of these communities have large populations of free-roaming domestic dogs and little access to veterinary care. These dogs have frequent contact with one another, canine feces, and a variety of refuse and foodstuffs that potentially contain zoonotic agents, all of which promote intestinal infection with a variety of zoonoses and subsequent human exposure.Despite the apparent zoonotic risks that domestic dogs may pose to indigenous Canadians, there are very few contemporary studies that characterize the microbial and parasite content of canine feces in these communities. This is problematic, because people infected with canine fecal zoonoses often exhibit non-specific clinical signs^6,8,9 that can be misdiagnosed if health care workers are unaware of the presence of these pathogens in their jurisdictions. Also, until the health risk posed by domestic dogs is better understood, it will not be possible to institute effective strategies to prevent human infection.It is also important to consider that exposure to canine fecal zoonoses could present a more significant health problem in indigenous communities compared with other Canadian populations. Indigenous peoples seem to be at increased risk for certain infectious diseases, including those caused by zoonotic pathogens,^5,7 likely because of traditional practices as well as risk factors associated with poverty, including poor nutrition and substandard housing.^5,10 Also, infectious diseases may have a more significant impact on the health of indigenous people compared with other Canadians because of concurrent health problems and decreased access to health care.¹⁰In 2008, a 6-year-old girl from a remote indigenous community in Northern Saskatchewan was diagnosed with an Echinococcus granulosus parasitic infection that was most likely acquired through contact with canine feces.¹¹ The ensuing, community-based epidemiologic investigation revealed widespread exposure to and infection with E. granulosus in humans and dogs, respectively,¹¹ creating concern that other zoonotic pathogens might be harbored by dogs in the community. To investigate this possibility, environmentally collected canine fecal samples were screened for a variety of bacterial and parasitic zoonoses.One block was randomly selected within each of the three distinct neighborhoods that comprise the main community. All yards on that block were surveyed on foot, and any canid feces found were collected in individual plastic bags. Fecal samples were also collected from around the community landfill based on the researchers'' suspicion that domestic dogs might frequent the landfill to scavenge on garbage. A total of 155 samples were collected from the four study sites. During the fecal-collection procedure, researchers observed numerous free-ranging dogs throughout the community, despite recent depopulation attempts. A numerical estimate of past or present dog populations could not be obtained.Samples were subdivided, and subsamples were sent to the World Health Organization Collaborating Center for the Molecular Epidemiology of Parasitic Infections (Murdoch University, Murdoch, Australia) and the University of Saskatchewan (Saskatoon, Canada) where they were analyzed for the presence of E. granulosus as previously described.¹¹At the University of Saskatchewan, subsamples were also analyzed using quantitative fecal-flotation¹² and sucrose-gradient¹³ techniques to concentrate and enumerate parasite eggs and Giardia spp. cysts/Cryptosporidium spp. oocysts, respectively.To determine the predominant Giardia spp. genotypes, polymerase chain reaction (PCR) was performed on selected samples as previously described¹⁴ to amplify a segment of the G. duodenalis β-giardin gene. Samples that contained > 10,000 Giardia spp. cysts/g were selected for analysis, because these feces had the potential to cause the greatest environmental contamination with Giardia spp. A total of 19 samples with > 10,000 cysts/g had sufficient material available for analysis. Four samples with 1,000–10,000 cysts/g were also tested to evaluate the sensitivity of the PCR assay at our institution. PCR was performed on DNA extracted from sucrose gradient concentrates using the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN Inc., Valencia, CA). Because G. duodenalis is the only Giardia spp. known to infect dogs^9,15 and all samples were observed to contain Giardia spp. cysts on microscopic examination, any failure to amplify product was interpreted to be the result of poor sample integrity or test sensitivity. Any product obtained by PCR was sequenced using the amplification primers. Sequencing of the β-giardin gene allows G. duodenalis samples to be classified into groups of genotypes called assemblages.¹⁴ This classification is essential when determining the zoonotic potential of Giardia spp. found in canine feces, because dogs may be infected with assemblages A, B, C, and D, of which only A and B are known to infect humans.^14–16 It should be noted that the genus Cryptosporidium contains several species with zoonotic potential.^15,16 The Cryptosporidium spp. identified in this study were not identified to the species level; however, domestic dogs have the potential to become infected with C. parvum and C. canis, both of which are known to cause disease in people.^15–17A subset of 60 fecal samples, which was comprised of 20 randomly selected samples from each of the three neighborhoods in the community, was selected for total bacterial DNA extraction (QIAGEN Stool Kit; QIAGEN Inc., Valencia, CA). These samples were tested for the presence of 14 known species of Campylobacter using a cpn60-based real-time quantitative PCR¹⁸ (also conducted at the University of Saskatchewan). Bacterial culture was not performed because of financial constraints and the degraded state of many of the samples.Distribution of pathogen-containing fecal samples and relative intensity of infection were compared among study sites using the χ², Wilcoxon rank sum, and Kruskall–Wallis tests.¹⁹ All calculations were performed using STATA/IC 10.0 (StatCorp LLP, College Station, TX) with a significance level of P < 0.05.Five genera of potentially zoonotic pathogens were found in 155 canine fecal samples collected within a northern Saskatchewan indigenous community (9^,14–16 Of the 60 samples tested for Campylobacter spp., 28 (47%) contained one or both of the established zoonotic species C. jejuni and/or C. upsaliensis.^8,20

Table 1

Prevalence and intensity of multiple zoonotic organisms identified in environmentally collected canine fecal samples from an indigenous Canadian community

HIV Protease Inhibitors, Indinavir or Nelfinavir, Augment Antimalarial Action of Artemisinin in vitro

Most malaria endemic regions are co-infested with HIV infection. Treatment of one may affect outcome of the other in co-infected individuals. HIV protease inhibitors, indinavir or nelfinavir, are important antiretroviral drugs and artemisinin is central to malaria treatment. We show these protease inhibitors augment the antimalarial activity of artemisinin against P. falciparum in vitro.Menace of malaria looms over one-fifth of the world population in more than 80 malaria endemic countries. Over one million people die annually due to Plasmodium falciparum malaria.¹ Most of these deaths occur in resource poor settings. The increased morbidity and mortality has been mainly due to widespread emergence of drug resistant P. falciparum strains. After the emergence of chloroquine resistance in P. falciparum, the only sure cure of malaria rests largely on artemisinin based combination therapies.¹ Artemisinin is now the only vital drug for effective treatment of malaria. Any resistance to it could be a fatal setback to malaria control programs. Recent appearance of artemisinin tolerant parasites at the Thai-Cambodia border is a cause of concern.^2,3 Most of the malaria-endemic countries are also burdened and co-infested by pandemic human immunodeficiency virus.^4,5 An estimated 40 million HIV-infected cases exist in Africa alone with an annual mortality of over 3 million people.⁶ Malaria is more prevalent, severe, and recurrent in HIV infected people than those without the virus.^4,7 This co-infection would also augment the spread of geographic boundaries of malaria infection in HIV prevalent areas.⁸ HIV infection is long lasting, requiring almost life long antiretroviral treatment, whereas malaria is self limiting. Treatment of one infection may affect outcome of the other in co-infected individuals. Novel antiretroviral treatments are being steadily introduced.⁹ The effect of HIV protease inhibitors on antimalarial potency of artemisinin has not been reported. This prompted us to evaluate the interaction of HIV protease inhibitors, indinavir or nelfinavir, on the antimalarial efficacy of artemisinin on chloroquine sensitive (3D7) and chloroquine resistant (RKL 303) strains of P. falciparum in vitro.Erythrocytic stages of chloroquine-sensitive P. falciparum, strain 3D7, and chloroquine-resistant RKL 303 strain were cultured and maintained as stocks on human B⁺ erythrocytes by candle-jar method¹⁰ at 37°C. Antimalarial potential of eight antiretroviral drugs (Matrix Laboratories Ltd., Secunderabad, India) and artemisinin (Sigma-Aldrich, St. Louis, MO) (11 with the aid of candle jar method.¹⁰ Synchronized ring stage parasites¹² were used in all the experiments. Percentage parasitemia and inhibition of parasite multiplication rate in relation to control was determined at the end of experiment by examining Giemsa-stained blood films. Counting of parasites was done in random adjacent microscopic fields, equivalent to about 3,000 erythrocytes at 1,000× magnification. The complete culture medium was prepared by adding sterile 5% sodium bicarbonate to Roswell Park Memorial Institute-1640 (Sigma-Aldrich) and supplemented with 10% B⁺ serum. Drug dilutions were prepared in antibiotic-free complete culture medium from the drug stock solution of 1 mg/mL in dimethyl sulfoxide (DMSO) 50% inhibitory concentration (IC₅₀) values were computed from log dose-response curve (Microsoft Excel, Microsoft Corp., Redmond, WA) and presented in 13 Each Shouldn''t there be more than one reference cited? “These in vitro results on inhibition of parasite multiplication rate by antiretroviral drugs reported here are comparable with those of others.^13”of these drugs was further investigated for its antimalarial interaction in combination with artemisinin in vitro using fixed ratio method. For combination assay, artemisinin was combined with either of the drugs in six fixed-ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5.^14,15 The first and the last ratio of these six preparations had the artemisinin and antiretroviral drug alone at a concentration approximately 8× their respective IC₅₀ value against a particular parasite strain. Further dilutions of these combinations have been described in plate preparation elsewhere.^14,15 After 48 hours, thin blood film slides from each triplicate experimental well and from control were prepared separately for counting the parasitemia. The fractional inhibitory concentration (FIC) for each drug in triplicate for six fixed-dose ratios was calculated by the following formula:

Table 1

Mean IC₅₀ values (± standard error) of artemisinin and antiretroviral drugs against Plasmodium falciparum (3D7 and RKL 303 strains) in vitro

Infectivity,Pathogenicity, and Virulence of Trypanosoma cruzi Isolates from Sylvatic Animals and Vectors,and Domestic Dogs from the United States in ICR Strain Mice and SD Strain Rats

Trypanosoma cruzi, the causative agent of Chagas disease, is widespread in the southern United States. In addition to detection in numerous wildlife host species, cases have been diagnosed in domestic dogs and humans. In the current investigation, groups of laboratory mice [Crl:CD1 (ICR)] were inoculated with one of 18 United States T. cruzi isolates obtained from a wide host range to elucidate their infectivity, pathogenicity, and virulence. In addition, laboratory rats (SD strain) were inoculated with four isolates. Mice and rats were susceptible to infection with all strains, but no morbidity or mortality was noted, which indicates that these T. cruzi isolates from the United States had low virulence for laboratory mice and rats.Trypanosoma cruzi, the causative agent of Chagas disease, infects approximately 10–12 million persons in the Americas; there are approximately 200,000 new cases annually.^1,2 In the United States, only six autochthonously acquired human infections have been reported. However, > 1,000 seropositive persons have been detected during routine screening of blood donations in the United States since 2007.³ Although few autochthonous human cases in the United States have been reported, reports of domestic dog and captive exotic animal cases are increasing,^4,5 and the prevalence of T. cruzi in wild mammal reservoir species can be as high as in South America.^6,7 Trypanosoma cruzi is currently categorized into one of six discrete typing units (TcI, TcIIa, TcIIb, TcIIc, TcIId, TcIIe). To date, all isolates from humans, vectors, wild mammals, domestic animals, and non-human primates in the United States have been classified as TcI or TcIIa.^8–10Identifying the genotype of a T. cruzi strain is often important for characterizing biological differences among isolates, such as virulence, pathogenicity, tissue tropism, geographic locality, and host/reservoir capacity. Previous mouse infection studies using several sylvatic- and domestic-derived isolates from Brazil showed that those from marsupials were generally more infective and generated higher parasitemias than those from vectors or placental mammals.^11,12 Patent infections were also more frequent in laboratory mice inoculated with TcII strains than TcI strains.¹² In contrast, U.S. isolates rarely cause morbidity and mortality in laboratory rodents,^13–20 but in one study, a T. cruzi isolate from a raccoon caused hind limb paralysis in mice.²¹Differences in infection outcome in these studies may be caused by different mouse strains, T. cruzi inoculum stage, inoculation route and/or dose, and source (host) species of the isolate. Additionally, many studies were conducted with genetically unclassified strains. The goal of the current study was to experimentally infect laboratory rodents with genetically classified T. cruzi isolates from the United States from a wide host range to determine infectivity, pathogenicity, and virulence. Based on previous studies on strains from the United States,^13–20 we hypothesized that sylvatic isolates would be infective but not virulent to mice.A total of 18 T. cruzi isolates from seven mammalian host species and two vector species was used in the study (​and2).2). These isolates were chosen to represent both genotypes (TcI and TcIIa) present in the United States and a diverse geographic and host range. Two isolates from Brazil (Y and Brazil strains) were used as positive controls (kindly provided by Dr. Rick Tarleton, University of Georgia, Athens, GA). Parasites stored in liquid nitrogen (first passage for all but the two Brazil strains) were rapidly thawed and established in DH82 canine macrophage monolayers to yield the infective culture–derived trypomastigotes.²²

Table 1

Detection of Trypanosoma cruzi in eight acutely-infected and one chronically-infected Crl:CD1 (ICR) mice^*