Mating, blood feeding, and oviposition of Simulium damnosum Theobald in the laboratory

The development of suitable techniques for colonizing the vectors of human onchocerciasis in the laboratory would facilitate critical studies on many aspects of their biology. Progress towards this end, however, has until now been prevented by the fact that no African simuliid had been induced to mate in captivity. The main vector of human onchocerciasis in Africa is Simulium damnosum, which exists in a number of different forms, some but not all of which bite man. During the present investigations one form of S. damnosum, the “Kibwezi” form, was successfully induced to mate, to blood-feed, and to lay viable eggs in the laboratory. The methods described should be tested on other forms of S. damnosum, especially the anthropophilic forms responsible for the transmission of Onchocerca volvulus.     

The distribution of the microfilariae of Onchocerca volvulus in the different body regions in relation to the attacking behaviour of Simulium damnosum s.l. in the Sudan savanna of northern Cameroon

Densities of Onchocerca volvulus microfilariae in four volunteers with low to moderate infections were estimated at five body sites by paired skin snips. The landing of Simulium damnosum s.1. females on the body of these volunteers was recorded during 12 hours for six days. Most flies fed at the ankles (53% and 51%) and calves (28% and 27% respectively) in both the standing and sitting positions. The density of microfilariae in the skin was highest in the pelvic region (24.1 mf/mg) and relatively low in the calf (14.8 mf/mg) and ankle (1.0 mf/mg) regions. From the biting rate (females/body part) and the microfilarial density (mf/mg) a transmission index was calculated for the different body regions. This was highest for the calves showing that this part of the leg, if unclothed, accounts for the highest rate of contact (50 to 60% of total) between vector and parasite.     

Optimal sample storage and extraction procotols for reliable multilocus genotyping of the human parasite Schistosoma mansoni

Genotyping individual larval stages and eggs of natural parasite populations is complicated by the difficulty of obtaining reliable genotypes from low quantity DNA template. A suitable storage and extraction protocol, together with a thorough quantification of genotyping errors are therefore crucial for molecular epidemiological studies. Here we test the robustness, handling time, ease of use, cost effectiveness and success rate of various fixation (Whatman FTA^® Classic and Elute Cards, 70% EtOH and RNAlater^®) and subsequent DNA extraction methods (commercial kits and proteinase K protocol). None of these methods require a cooling chain and are therefore suitable for field collection. Based on a multiplex microsatellite PCR with nine loci the success and reliability of each technique is evaluated by the proportion of samples with at least eight scored loci and the proportion of genotyping errors. If only the former is taken into account, FTA^® Elute is recommended (83% success; 44% genotyping error; 0.2€/sample; 1 h 20 m handling time). However, when also considering the genotyping errors, handling time and ease of use, we opt for 70% EtOH with the 96-well plate technology followed by a simple proteinase K extraction (73% success; 0% genotyping error; 0.2€/sample; 15 m handling time). For eggs we suggest (1) to pool all eggs per person in 1.5 ml tubes filled with 70% EtOH for transport and (2) to identify each egg to species level prior to genotyping. To this end we extended the Rapid diagnostic PCR developed by Webster et al. (2010) with a S. mansoni-specific primer to discriminate between S. mansoni, S. haematobium and S. bovis in a single PCR reaction. The success rate of genotyping eggs was 75% (0% genotyping error). This is the first study to incorporate genotyping errors through re-amplification for the evaluation of schistosome sampling protocols and the identification of error-prone loci.     

DNA immunisation with Onchocerca volvulus chitinase induces partial protection against challenge infection with L3 larvae in mice

The parasitic nematode, Onchocerca volvulus is a major cause of blindness and dermal pathology in tropical regions. A vaccine directed to infective larvae would provide a valuable control tool alongside the current methods of chemotherapy and vector control. Previously we have described the identification of a chitinase cDNA that is expressed in a stage specific manner by O. volvulus infective third stage (L3) larvae. To evaluate its host protective potential, the complete open reading frame was cloned into the eukaryotic expression plasmid pJW4303 and used to vaccinate mice by DNA immunisation with the Accell GeneGun. The survival of challenge infective larvae was monitored using implanted micropore chambers. In the first trial, mice immunised 3 times over 4 months with 1 μg O. volvulus chitinase DNA responded with modest antibody responses dominated by IgG2a and exhibited a 36% (p=0.189, NS) reduction in parasite survival compared with challenge controls. In the second trial, an increased dose of DNA (5 μg) and more frequent immunisations (5 times over 6 months) stimulated an IgG1 dominant response and a 53% reduction in parasite survival (p=0.042). Antibodies from the vaccinated mice reacted with the cuticle of post-infective L3 larvae, implying that this may be the site of immune attack following secretion of chitinase.     

Comparison of mounting methods for the evaluation of fibers by phase contrast microscopy

The objectives of this study were to evaluate mounting methods for fiber examination of air sample filters by phase contrast microscopy (PCM) and to evaluate differences in fiber counts that might be due to fiber movement. Acetone/triacetin (AT) with various amounts of triacetin and acetone/Euparal (AE) where the mounting medium was placed between the cleared filter wedge and the coverslip were tested as a function of time. Field sample slides collected from a taconite iron-ore processing mill, a tremolitic talc-ore processing mill, and from around a crusher in a meta-basalt stone quarry were prepared with relocatable coverslips to revisit the same field areas on the slides. For each slide, three or four field areas were randomly selected and pictures were taken every 2 weeks to determine any sign of fiber movement over time. For 11 AT slides (named as AT-3.5) prepared with 3.5 μl of the mounting medium according to the NIOSH 7400 method, no fiber movements were detected over 59 weeks. On the other hand, AT slides prepared with larger quantities (10, 15, and 20 μl) of the mounting medium (named as AT-10) and AE slides prepared with ～10 μl mounting medium showed fiber movement from the eighth day at the earliest. Fiber movement began earlier for the slides mounted with excess triacetin than for those mounted with Euparal. The sample slide storage method, either vertically or horizontally, did not seem to accelerate fiber movement. Additionally, two other modified methods, dimethylformamide solution/Euparal (mDE) and dimethylformamide solution/triacetin (mDT), were also prepared where the mounting medium was placed between the cleared filter wedge and the glass slide. The findings of fiber movements were similar; when 3.5 μl of triacetin was used for the mDT slides, fiber movements were not detected, while fibers on slides prepared with 10 μl triacetin (mDT-10) moved around. No fiber movements were observed for the mDE slides at any time during 59 weeks. Once fiber movement started, fibers moved over distances measured from 4 μm and up to >1000 μm within 22 weeks. However, since then, no further fiber movements have been observed in any field sample slides. Additional sample slides, two Amosite and two chrysotile, were prepared from Union for International Cancer Control (UICC) samples using the AT method with 5 μl triacetin mounting medium. Fiber movements were also observed in these samples; chrysotile fibers began to migrate in 3 weeks, while Amosite fiber movement started after 3 months. Although fiber movement was observed for the AT-10, AE, and mDT-10 sample slides, fiber counts were not significantly different from AT-3.5 and mDE samples that exhibited no fiber movement. Although fiber counts would not be significantly changed by fiber movement, the type and amount of mounting medium for sample slide preparation remains critical for issues such as quality assurance and training of analysts by revisiting the same fibers.     

HIV检测基因芯片的研制和应用评价

目的研制用于HIV检测的基因芯片,并评价其在临床诊断和出入境检疫中的应用价值。方法针对HIV-1gags基因相对保守区域,设计检测探针,并设计内标和对照探针,制备HIV检测微阵列基因芯片;设计引物,以1:20引物比例不对称扩增HIV基因靶标,Cy3荧光标记扩增产物,作为杂交模板。Cy3荧光标记PCR扩增产物与微阵列探针杂交反应,结果经荧光扫描,并用分型软件分析判断阳性探针和样本HIV阴阳性。采用信号放大、梯度临界值技术,增加了检测的灵敏度和特异性。采用70例临床样本实样检测与DNA测序作对照,评价试剂盒的性能和应用价值。结果70例样本,采用本试剂盒与DNA测序并行检测,两者符合率为98.57%。结论本HIV检测基因芯片试剂盒具有高通量、操作简便、低成本、准确度好等优点,应用研究表明,在临床HIV诊断检测和出入境检测方面具有很高的应用价值。     

Morphometric differentiation of Onchocerca volvulus and O. ochengi infective larvae.

By microinjection of cryopreserved microfilariae (mf) into nulliparous flies, a comparison of the lengths of the infective larvae (L3) of Onchocerca volvulus and O. ochengi from the head of Simulium damnosum s.l. (presumed S. sirbanum) has been made. The suitability of S. sirbanum as a host was similar for both Onchocerca spp. The mean length +/- standard deviation of O. ochengi infective larvae measured in aqueous medium after storage of infected flies in liquid nitrogen was 762 +/- 63 microns (n = 39), significantly longer (P much less than 0.0001) than those of a savanna isolate of O. volvulus (676 +/- 56 microns, n = 26). Although the frequency distributions of the lengths of larvae of the 2 species overlapped, a critical value for discrimination of 719 microns applied to normally distributed populations with means and standard deviations of these samples would result in correct classification of 78% of true O. volvulus and 75% of true O. ochengi. A discriminant function analysis incorporating width measurements did not usefully improve the level of accuracy of discrimination. Larvae from flies stored in 70% ethanol and stained with acid haemalum were about 10% shorter, but O. ochengi infective larvae were still proportionately longer than those of O. volvulus (693 +/- 40 microns, n = 45 compared to 580 +/- 38 microns, n = 6, respectively). These data show that the infective L3 of O. volvulus and O. ochengi differ morphologically. Although the population length distributions overlap, by classifying larvae greater than 719 microns long as O. ochengi and those less than 719 microns long as O. volvulus a more accurate estimation of true O. volvulus infection rates in S. damnosum s.l. can be derived than is currently possible.     

Detection of Cryptosporidium parvum oocysts in experimentally contaminated lettuce using filtration, immunomagnetic separation, light microscopy, and PCR

Recovery of Cryptosporidium parvum oocysts in a fecal suspension that experimentally contaminated onto lettuce leaves was investigated. Material recovered from the lettuce samples by washing in detergent solutions were concentrated by filtration using the Envirochek Sampling Capsule. Oocysts were concentrated by immunomagnetic separation (IMS) and detected by microscopy following modified Ziehl-Neelsen (MZN) staining. Cryptosporidal DNA was detected using a nested-PCR assay for amplification of a fragment of the Cryptosporidium oocyst wall protein (COWP) gene, which was applied to DNA extracted from both filtrates, and material recovered from MZN stained smears on glass slides after microscopy. No Cryptosporidium were detected by microscopy or by PCR of un-inoculated lettuce leaves. After IMS, means of 0-6.5% of the total numbers of oocysts inoculated were recovered and detected by microscopy. Detection by PCR was less sensitive than microscopy. There was a strong association between successful PCR amplification, the numbers of oocysts detected by microscopy and the numbers of oocysts in the inoculum. This study confirms that C. parvum oocysts can be recovered from contaminated lettuce using filtration and IMS, and detected by microscopy and PCR. However, further developments are required to improve recovery of this parasite.     

新型冠状病毒核酸检测样本DNA污染鉴别方法研究

目的 采用不同实时荧光PCR方法检测各类型DNA污染模拟样本,分析检测方法性能,为在新型冠状病毒(简称新冠病毒)核酸检测中鉴别DNA污染提供依据。 方法 取实验室保存的新冠病毒RNA核酸样本和新冠病毒阳性质控品(含病毒序列的质粒样本),按不同比例混合,并进行梯度稀释,制成模拟样本。分别使用实时荧光RT-PCR、灭活逆转录酶前加样并去除RT步骤的PCR(PCR Set 1)和灭活逆转录酶后加样并去除RT和灭活步骤的PCR(PCR Set 2)三种不同方法,使用同一型号设备对同一批模拟样本进行检测。记录并分析检测结果,确定最佳的DNA污染判断方法。 结果 新冠病毒RNA核酸样本中,相比实时荧光RT-PCR,PCR Set 1下所有样本的Ct值增高2-4个循环,PCR Set 2中除原始样本N基因阳性外,均为阴性。新冠病毒DNA污染样本三种方法检测Ct值无明显差异。含等量DNA污染的混合污染样本和含梯度DNA污染的混合污染样本三种方法检测显示Ct值存在差异。三种方法检测不同新冠病毒基因,变化规律无明显差异。 结论 PCR Set 1和PCR Set 2两种方法均可用于鉴别DNA污染,特别是PCR Set 2方法对各浓度的DNA污染均有较好的区分能力。     

Molecular studies of Onchocerca volvulus isolates from Mexico.

DNA from Onchocerca volvulus from Oaxaca and Chiapas, Mexico were used as templates to amplify members of the O-150 Onchocerca specific repeat sequence family. The resulting PCR amplicons all hybridized with OVS2, an oligonucleotide that has been previously shown to recognize amplicons derived from O. volvulus with 100% sensitivity. However, when PCR products amplified from the O. volvulus specific plasmid pOVS134 were used as a probe, most samples did not hybridize. Similarly, when PCR products amplified from DNA isolated from adult O. volvulus from Oaxaca were used as a probe, amplicons from adult worms from both Oaxaca and Chiapas were recognized, but PCR products from infected black flies from Chiapas were not recognized. Amplicons derived from an adult worm from Chiapas hybridized with PCR products produced from adult parasites from both Oaxaca and Chiapas and to PCR products derived from the DNA of infected black flies from Chiapas. These data, when taken together, suggest that differences exist among the repeat sequence populations of parasites from Oaxaca and Chiapas in Mexico, suggesting that the O-150 repeat sequence family may be a useful tool for biogeographic studies of O. volvulus in the Americas.     

Standardization of conditions for PCR detection of Leishmania spp. DNA in sand flies (Diptera, Psychodidae)

The correct identification of etiological agents in vector insects is crucial for epidemiological studies. Identification of flagellates in such vectors, usually by dissection of the digestive tract and microscopic observation of the contents as well as attempts at parasite isolation from insects in culture media, have proven operationally inadequate and with poor diagnostic specificity, since female sand flies are also hosts for other flagellates like Trypanosoma and Endotrypanum. Due to the efficiency and specificity of DNA target sequence amplification by polymerase chain reaction (PCR), the latter could be used to investigate the presence of Leishmania in sand flies, although the insects need to be properly stored and the Leishmania DNA extracted using appropriate methodology. This paper describes methodologies to standardize sand fly storage and Leishmania DNA extraction in such specimens as a more practical method in field studies.     

High levels of Trypanosoma cruzi DNA determined by qPCR and infectiousness to Triatoma infestans support dogs and cats are major sources of parasites for domestic transmission

The competence of reservoir hosts of vector-borne pathogens is directly linked to its capacity to infect the vector. Domestic dogs and cats are major domestic reservoir hosts of Trypanosoma cruzi, and exhibit a much higher infectiousness to triatomines than seropositive humans. We quantified the concentration of T. cruzi DNA in the peripheral blood of naturally-infected dogs and cats (a surrogate of intensity of parasitemia), and evaluated its association with infectiousness to the vector in a high-risk area of the Argentinean Chaco. To measure infectiousness, 44 infected dogs and 15 infected cats were each exposed to xenodiagnosis with 10–20 uninfected, laboratory-reared Triatoma infestans that blood-fed to repletion and were later individually examined for infection by optical microscopy. Parasite DNA concentration (expressed as equivalent amounts of parasite DNA per mL, Pe/mL) was estimated by real-time PCR amplification of the nuclear satellite DNA. Infectiousness increased steeply with parasite DNA concentration both in dogs and cats. Neither the median parasite load nor the mean infectiousness differed significantly between dogs (8.1 Pe/mL and 48%) and cats (9.7 Pe/mL and 44%), respectively. The infectiousness of dogs was positively and significantly associated with parasite load and an index of the host’s body condition, but not with dog’s age, parasite discrete typing unit and exposure to infected bugs in a random-effects multiple logistic regression model. Real-time PCR was more sensitive and less time-consuming than xenodiagnosis, and in conjunction with the body condition index, may be used to identify highly infectious hosts and implement novel control strategies.     

Comparison of Three Methods for Diagnosis of Cutaneous Leishmaniasis

Background

Leishmaniasis is one of the infectious parasitic diseases of highest incidence in the world. Cutaneous Leishmaniasis (CL) has long been reported in Shiraz, Southern Iran. There is a need to find a sensitive and specific method for treatment and control of the disease.

Methods

We have compared the sensitivity of the conventional methods microscopy and cultivation of lesion scrapes against PCR amplification of parasite kinetoplast DNA from these samples. The samples (n=219) were obtained from the patients clinically suspected of CL. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal (NNN) blood agar for promastigote growth. For PCR, the dry smears were scraped off the slides and DNA was extracted.

Results

The positive rates from 219 specimens were 76.71%, 50.68%, and 93.61% for microscopy, cultivation, and PCR, respectively. The highest correlation was found between PCR and microscopy method (P=0.014). In PCR assay, 95.61%, 3.9%, and 0.49% of the samples were identified as Leishmania major, L. tropica, and dermatropic L. infantum, respectively.

Conclusion

The PCR method appears to be the most sensitive for the diagnosis of CL and is valuable for identifying the other species of Leishmania with confusing dermatropic signs.     

Comparison of methods for collection of DNA samples by mail in the Black Women's Health Study

PURPOSE: The authors compared approaches to participants and methods of collecting buccal cell samples by mail in the Black Women's Health Study, a follow-up study of geographically dispersed African-American women. Outcomes of interest were within group participation rates, yield of DNA, and PCR success. METHODS: Six hundred fifty six participants were randomized to four groups: Groups 1 and 2 used the cheek swab method and Groups 3 and 4 used the mouthwash swish method. Groups 1 and 3 were mailed collection kits together with consent forms, whereas Groups 2 and 4 were mailed a kit only after returning a signed consent. RESULTS: Participation rates were similar regardless of the method used for sample collection or mailing of the kits: samples were returned by 40% of Group 1, 37% of Group 2, 36% of Group 3, and 35% of Group 4. The median DNA yield was 3693 ng/sample for the swab method and 10,077 ng/sample for the mouthwash swish method (p<0.001). PCR analyses were successful in 98% of mouthwash samples and 94% of swab samples. CONCLUSION: Because of its higher yield of DNA, the mouthwash swish method is preferable for collection of buccal cell samples by mail.     

Susceptibility of Simulium damnosum complex larvae to temephos in the Tukuyu onchocerciasis focus, southwest Tanzania

Tukuyu onchocerciasis focus was earmarked for vector control using insecticide against larval stages. Susceptibility tests of mature larvae of Simulium damnosum s.l. vectors to temephos insecticide were carried out before and after two years of insecticide treatment of rivers within Tukuyu onchocerciasis focus, south-western Tanzania. The tests were done in 1999/2000 and 2004 using WHO standard methods. Mature larvae were exposed to 9 concentrations of temephos active ingredient, from the weakest 0.00975mg/litre to the strongest of 2.5mg/l. Each test concentration and control was run in duplicates of 25 larvae each, set for three hours in a cool temperature. After incubation, test solution was discarded and larval condition checked. Numbers of larvae in each category were recorded and used to determine mortality rate for each concentration as well as for the LC50 and LC95. A total of 1,666 larvae were tested, 942 during the pre- and 724 post-treatment. Results showed that both pre and post-treatment samples were susceptible, attaining 100% mortality at the diagnostic dose of 1.25mg/l, and LC50 between 0.129-0.34mg/l pre - and 0.144-0.211 mg/l (95% CI, P < 0.05) post- treatment. These values fall within the standard diagnostic dose of < or = 0.4mg/l for susceptible S. damnosum s.l populations. It was concluded that the endemic S. damnosum population was susceptible to temephos before and after two years of intermittent field application. Temephos was thus recommended for continued use in onchocerciasis vector control in the Tukuyu focus, to complement Community Directed Treatment with Ivermectin, but close monitoring of vector susceptibility should be done.     

Digital storage of glass slides for quality assurance in histopathology and cytopathology

Proficiency testing programmes for measuring screening skills in pathology are mainly conducted using conventional glass microscope slides. However, the availability of robotic microscopes allows an entire conventional slide to be digitized. Our experiments have shown that, using a widely available robotized microscope and a PC, the image of a single field may be acquired in 2 s on average, including stage movements, autofocus and storage. Digitizing an entire slide, a fully automated procedure, takes up to 8 h. If the image of each field is compressed at an appropriate quality level (a compression ratio of, say, 35:1) it requires about 40 kByte to be stored, resulting in a total storage requirement of about 600 MByte per slide. Thus one CD-ROM can be used to store one virtual slide, as well as a self-installing program to provide a microscope simulator facility. This allows pathologists to examine the virtual case from their computer in a similar manner to looking at a glass slide on a conventional microscope. This permits a new, computer-based approach to proficiency testing in histopathology and cytopathology. Use of virtual slides should encourage the diffusion of national quality assurance programmes, which at present suffer from certain organizational and logistical limitations.     

A quantitative polymerase chain reaction method for the detection in avian faeces of salmonellas carrying the spvR gene.

A quick, semi-quantitative method of detecting Salmonella species which contain the virulence plasmid has been developed using the polymerase chain reaction (PCR). A pair of primers have been synthesized encompassing a 500 bp fragment of the spvR virulence gene. Competitor DNA consisting of the spvR gene with a 94 bp deletion situated between the primer recognition sequences, was cloned into a plasmid vector. Co-amplification of the ''unknown'' target salmonella DNA with known quantities of competitor DNA in the same reaction tube gave PCR products of 500 and 406 bp respectively. Visual assessment of the ratio of the two products on ethidium bromide stained agarose gels provided an estimate of the approximate number of salmonella cells present in avian faeces. The technique could be applied to detect quantifiably any non-host DNA in clinical samples if a suitable DNA sequence for primer construction is available.     

pcDNA3．1（＋）／NspA真核表达载体的构建及鉴定

目的构建pcDNA3.1( )淋病奈瑟菌外膜蛋白—奈瑟菌表面蛋白A(Neisseria surface Protein A,NspA)基因真核表达载体,为研制淋病奈瑟菌核酸疫苗奠定基础。方法根据淋病奈瑟菌NspA基因序列,设计合成一对引物,用PCR方法从淋病奈瑟菌基因组DNA中扩增出NspA基因片段,将扩增的产物连接于测序载体pUCm-T上,并构建重组体pcDNA3.1( )/NspA,进行酶切、PCR及测序鉴定。结果NspA基因体外扩增产物大小约为525 bp。所构建的pcDNA3.1( )/NspA重组体经双酶切及PCR鉴定,与预期片断的大小相符;测序结果与GenBank中收录的NspA全长序列一致,表明pcDNA3.1( )/NspA真核表达载体构建正确。结论成功地构建了淋病奈瑟菌真核重组表达载体pcD-NA3.1( )/NspA,为NspA蛋白的功能研究和淋病奈瑟菌核酸疫苗的研制提供了物质基础。     

Methods for dissecting dry insects and insects preserved in fixative solutions or by refrigeration

The methods described in this paper for the dissection of dry and preserved insects have been used for several years on various species, mainly mosquitos. In the past, dry or partially dry mosquitos found in traps or in the laboratory had to be discarded. By softening these insects in a detergent solution, however, it is possible to make most observations in the same way as on fresh material. The preservation of insects in the dry state, in a fixative, or in the refrigerator after collection enables much larger samples to be studied; the whole of the material can be examined and the work can be done when time permits. In addition, material can be sent to central laboratories far from the place of collection, and infected insects can be kept in stock for teaching purposes.