Buffy Coat Microscopy Research Articles

African Animal Trypanosomiasis: A Systematic Review on Prevalence, Risk Factors and Drug Resistance in Sub-Saharan Africa.

African animal trypanosomiasis (AAT) a parasitic disease of livestock in sub-Saharan Africa causing tremendous loses. Sub-Saharan continental estimation of mean prevalence in both large and small domestic animals, risk factors, tsetse and non-tsetse prevalence and drug resistance is lacking. A review and meta-analysis was done to better comprehend changes in AAT prevalence and drug resistance. Publish/Perish software was used to search and extract peer-reviewed articles in Google scholar, PubMed and CrossRef. In addition, ResearchGate and African Journals Online (AJOL) were used. Screening and selection of articles from 2000-2021 was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Articles 304 were retrieved; on domestic animals 192, tsetse and non-tsetse vectors 44, risk factors 49 and trypanocidal drug resistance 30. Prevalence varied by, host animals in different countries, diagnostic methods and species of Trypanosoma. Cattle had the highest prevalence with Ethiopia and Nigeria leading, T. congolense (11.80-13.40%) and T. vivax (10.50-18.80%) being detected most. This was followed by camels and pigs. Common diagnostic method used was buffy coat microscopy. However; polymerase chain reaction (PCR), CATT and ELISA had higher detection rates. G. pallidipes caused most infections in Eastern regions while G. palpalis followed by G. mortisans in Western Africa. Eastern Africa reported more non-tsetse biting flies with Stomoxys leading. Common risk factors were, body conditions, breed type, age, sex and seasons. Ethiopia and Nigeria had the highest trypanocidal resistance 30.00-35.00% and highest AAT prevalence. Isometamidium and diminazene showed more resistance with T. congolense being most resistant species 11.00-83.00%.

Bovine trypanosomosis: Prevalence and vector distribution in Sadi Chanka district, Kellem Wollega zone, Oromia regional state, Ethiopia

Bovine trypanosomosis remains an important livestock disease constraint, which is threatening livestock health and production, despite ongoing tsetse and trypanosomosis control efforts in Sadi Chanka district, Kellem Wollega zone, Oromia regional state, Ethiopia. A cross-sectional study was conducted in May 2018, to determine the prevalence of bovine trypanosomosis and distribution of the vectors of disease in Sadi Chanka district, Western Ethiopia. A total of 370 blood samples were collected from randomly selected local Horro and Abigar cattle breeds covering five villages of the district. The collected samples were examined using buffy coat microscopy and Giemsa-stained thin blood smear techniques. In this study, 12.4% (95% CI: 12.3-12.4) of the animals were found to be infected with trypanosomes. The study showed that 69.6% of trypanosome infections were caused by T. congolense followed by 26.1% T. vivax and 4.3% mixed T. congolense and T. vivax. In the present study, the association of bovine trypanosomosis was assessed in releation to body condition scores, sex, and age of cattle, and a significant association (P<0.05) was observed between body condition scores. However, significant differences were not observed between sex and age categories (P>0.05). The Mean Packed Cell Volume (PCV) of infected (21.6%) and non-infected (24.5%) groups of cattle had significant variation (P<0.05). In an entomological survey, a total of 616 flies were trapped, of which 280 (45.5%) were Glossina and the remaining 336 (54.5%) were Stomoxys, Tabanus, and Haematopota. The apparent density of Glossina, Stomoxys, Tabanus, and Haematopota was 3.5, 3.1, 0.7 and 0.4 fly per trap per day, respectively. This study generated basic scientific data on the epidemiology of bovine trypanosomosis and its vectors in Sadi Chanka district, which can be used in planning the control of bovine trypanosomosis in the area.

Risk-free point-of-care visceral leishmaniasis diagnostics: combining buffy coat microscopy and immunoassay in tertiary rural hospitals in Sudan

Visceral Leishmaniasis (VL), a life-threating disease in Sudan and Eastern Africa, is usually diagnosed by a painful and invasive tissue aspirate microscopy. This study assessed the diagnostic effectiveness of buffy coat (BC) microscopy and the rK39 immunoassay test separately and combined as an easy non-invasive method applied to peripheral blood sample for field diagnosis of VL. 151 VL suspected patients were recruited from tertiary rural hospitals in Bazura, Gedaref state, from 2014-2015. All patients were tested for VL using rK39 ICT and microscopy smears from LN aspirate and BC in addition to PCR from BC as a reference standard test. Both BC and LN aspirate microscopy showed perfect specificity (100%) with false negative results, while the majority of true positives (81%) had a low-parasite burden. ICT showed almost perfect agreement but limited by its poor specificity. Each of these three tests is inadequate as a consistent single diagnostic tool and should be replaced by PCR in routine practice. Combining the results of risk-free BC microscopy and rk39 ICT, using peripheral blood sample, improved VL diagnosis with almost perfect agreement and 93.4% accuracy. Our findings indicate that combined BC microscopy and ICT are accurate, simple and easy point-of-care VL diagnostic tools in community and rural hospitals that can replace or reduce the use of invasive tissue aspirates microscopy, when PCR is unavailable. This is particularly of value in endemic rural areas, decreasing the delay in final diagnosis and preventing deaths caused by VL.

Multiplex real-time PCR for diagnosing malaria in a non-endemic setting: a prospective comparison to conventional methods

Almost a decade ago our diagnostic laboratory implemented an in-house real-time PCR for the detection of Plasmodium DNA to diagnose malaria in parallel with conventional diagnostics, i.e., microscopy (thick and thin smears), quantitative buffy coat microscopy (QBC), and a rapid diagnostic test (RDT). Here we report our experiences and make a comparison between the different diagnostic procedures used in this non-endemic setting. All patients during the period February 2009–December 2017 suspected of malaria were prospectively tested at the moment of sample collection. Both PCR and conventional malaria diagnostics were carried out on a total of 839 specimens from 825 patients. In addition, three Plasmodium falciparum (Pf) patients were closely followed by real-time PCR and microscopy after treatment. Overall, 56 samples (55 patients) tested positive by real-time PCR, of which six were missed by microscopy and seven by QBC. RDT showed fairly good results in detecting Pf, whereas specificity was not optimal. RDT failed to detect 10 of 17 non-Pf PCR positive specimens. One Plasmodium malariae patient would have been missed if only conventional diagnostic tests had been used. The high sensitivity of the PCR was confirmed by the number of PCR positive, microscopy negative post-treatment samples. In conclusion, within our routine diagnostic setting, malaria real-time PCR not only showed a high level of agreement with the conventional methods used, but also showed higher sensitivity and better specificity. Still, for complete replacement of the conventional procedures in a non-endemic setting, the time-to-results of the real-time PCR is currently too long.

New molecular detection methods of malaria parasites with multiple genes from genomes

For the effective control of malaria, development of sensitive, accurate and rapid tool to diagnose and manage the disease is essential. In humans subjects, the severe form of malaria is caused by Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) and there is need to identify these parasites in acute, chronic and latent (during and post-infection) stages of the disease. In this study, we report a species specific and sensitive diagnostic method for the detection of Pf and Pv in humans. First, we identified intra and intergenic multiloci short stretch of 152 (PfMLS152) and 110 (PvMLS110) nucleotides which is present up to 44 and 34 times in the genomes of Pf and Pv respectively. We developed the single-step amplification-based method using isolated DNA or from lysed red blood cells for the detection of the two malaria parasites. The limit of detection of real-time polymerase chain reaction based assays were 0.1copyof parasite/μl for PfMLS152 and PvMLS110 target sequences. Next, we have tested 250 clinically suspected cases of malaria to validate the method. Sensitivity and specificity for both targets were 100% compared to the quantitative buffy coat microscopy analysis and real-time PCR (Pf-chloroquine resistance transporter (PfCRT) and Pv-lactate dehydrogenase (PvLDH)) based assays. The sensitivity of microscopy and real-time PCR (PfCRT and PvLDH primers) assays were 80.63%; 95%CI 75.22%–85.31%; p<0.05 and 97.61%; 95%CI 94.50%–99.21%; p<0.05 in detecting malaria infection respectively when compared to PfMLS152 and PvMLS110 targets to identify malaria infection in patients. These improved assays may have potential applications in evaluating malaria in asymptomatic patients, treatment, blood donors and in vaccine studies.

Failure of imidocarb dipropionate to eliminate Hepatozoon canis in naturally infected dogs based on parasitological and molecular evaluation methods

The efficacy of imidocarb dipropionate for the treatment of Hepatozoon canis infection was studied in three naturally infected asymptomatic dogs followed longitudinally over 8 months. Response to treatment was followed by monitoring blood counts, parasitemia levels in blood, parasite in concentrated buffy-coat smears and by PCR. The dogs were initially treated with a low dose of 3 mg/kg imidocarb dipropionate twice a month and when parasitemia persisted after five treatments, with the regular dose of 6 mg/kg. In one dog, H. canis gamonts were no longer detectable by blood and buffy-coat microscopy after 2 months of therapy with 6 mg/kg while in the two other dogs gamonts were intermittently found in blood but persistently detectable in buffy-coat smears during the whole study period. Furthermore, combined therapy with doxycycline monohydrate administered at 10 mg/kg/day PO for 4 weeks also failed to eliminate H. canis. PCR revealed that parasite DNA was present in the blood of all dogs at all sampling dates regardless of treatment refuting the effectiveness of treatment suggested by negative blood microscopy. Detection of H. canis in buffy coat was found to be twice as sensitive than by blood smear and detection by PCR was even more sensitive revealing infection in eight samples (16% of total samples) negative by blood and buffy-coat microscopy. In conclusion, imidocarb dipropionate was not effective in eliminating H. canis from dogs treated repeatedly over 8 months. Microscopical detection is not sufficient for the evaluation of treatment response in H. canis infection and follow up by molecular techniques is recommended.

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique.

We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n = 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.

Early diagnosis of septicaemia in infants with respiratory distress in the tropics.

A prospective study of buffy coat microscopy of 108 infants with respiratory distress during the first 48 h of life was undertaken. Thirty-two infants (29.6%)had positive blood cultures. The buffy coat was stained with methylene blue and with Gram's stain. Using methylene blue staining, 94% shared organisms in the buffy coat. Whereas using Gram's stain only 50% were positive--all of which were also positive with methylene blue. Twenty-four infants with negative blood cultures had radiological signs of pneumonia. Of these, 83% showed organisms in the buffy coat with both methylene blue and Gram staining. We conclude that buffy coat microscopy provides a simple and reliable guide in the early diagnosis of neonatal septicaemia or lung infection in infants with respiratory distress.

A silicone centrifugation technique for the detection of low parasitaemias of salivarian trypanosomes

A method using silicone fluid of specific gravity 1·075 was employed to detect low numbers of salivarian trypanosomes in rats infected with T. brucei, T. gambiense, T. congolense or mouse-adapted T. vivax. This method compared favourably with other microsensitive techniques such as the miniature anionexchange centrifugation and microhaematocrit buffycoat microscopy methods. The silicone centrifugation technique is based on the density differences between the host's erythrocytes and the parasites. Under the conditions used, the red cells are pelleted by centrifugation through a layer of silicone fluid whereas the trypanosomes remain in the plasma supernatant.

Field diagnosis of sleeping sickness in the Ivory Coast. I. Comparison of the miniature anion-exchange/centrifugation technique with other protozoological methods

The miniature anion-exchange/centrifugation (m-AEC) technique for the detection of trypanosomes in blood was tried out as a field diagnostic method for African sleeping sickness among populations exposed to natural infection in the vicinity of Daloa, République de la Côte d'Ivoire. It was compared with the two other protozoological techniques in common use in Africa, namely, the microhaematocrit buffy-coat microscopy (MBCM) method and the thick blood film (TBF). A laboratory study on a small number of known infected subjects and two field-survey studies, all showed the m-AEC method to be more sensitive than the MBCM and very much more sensitive than the TBF. The m-AEC method detected many cases that were missed by one or other, or both, of the other tests. The m-AEC method has, also, other advantages. The duration of critical microscopy required is limited. It is faster in operation when large numbers of subjects are concerned than is the MBCM. It offers, with no additional operation beyond filling the eluates into suitable tubes, samples of diluted plasma which may be used for serology. Thus a very efficient survey procedure can be envisaged: first the m-AEC test, selecting infected subjects for immediate treatment; second, re-examining, by the m-AEC method, on a subsequent visit, those other subjects suspect on the basis of the serological examination of the eluates. The complexities of the preparation of materials and procedures, which hitherto have inhibited general application in the field of techniques involving anion-exchange, have been avoided by miniaturization and by the pre-preparation of apparatus and materials at a base laboratory.

Trypanosoma brucei: miniature anion-exchange centrifugation technique for detection of low parasitaemias: adaptation for field use

The miniature anion-exchange/centrifugation (AEC) method, originally developed for the detection of submicroscopic trypanosomaemias in laboratory rodents, has been adapted for the diagnosis of trypanosomiasis in man in the field using blood samples obtained by finger-prick. It has been tested in a survey in The Gambia. The method is shown to be highly sensitive and to fulfil the first essential criteria for exploitation in the field, namely, that it can be operated in the open air under tropical conditions, and that an adequate number of subjects can be examined in a normal working day at an acceptable cost. The method also offers two advantages over the other highly sensitive method applicable to small blood samples, the microhaematocrit buffy-coat microscopy (MBCM) method, namely, that it minimizes the requirements for highly critical microscopy and provides, in the same operation, samples of diluted plasma which can be used for serological study.