Quantitative Buffy Coat Technique Research Articles

Misidentification of Plasmodium Species by Cross-Reacting Primers and Cerebral Malaria Caused by Plasmodium vivax

Abstract Introduction The clinical presentation of a case as cerebral malaria with molecular identification confirming it as Plasmodium vivax underlines the importance of using molecular tools to identify the species and type of malaria. The possibility of the relationship between the complication observed during clinical diagnosis and the multifactorial molecular changes could likely be the reason for terming it cerebral malaria. Methods We report four cases analyzed using the quantitative buffy coat technique followed by classical Giemsa stained thick-film microscopy, and nested polymerase chain reaction for the genus-specific region of Plasmodium targeting 18S rDNA followed by species-specific identification with a different set of primers and products confirmation with sequencing. Results Primers targeting P. knowlesi generated the expected product size of 153 base pairs that, upon sequencing, matched with the P. vivax sequence reflecting the relatedness of the species. Likewise, primers targeting P. ovale generated a 456 product whose sequence matched the P. vivax sequence. Conclusion Infection with P. vivax can potentially cause cerebral malaria, and P. vivax can cause severe malaria complications alone or mixed with other species and can show cerebral malaria signs, which are typically associated with P. falciparum infections. The sequence relatedness reflects the genome similarity between P. knowlesi and P. ovale with P. vivax. The need to reconfirm with an additional set of newly reported primers is mandatory.

Diagnosis, Clinical and Molecular Delineation of Human Plasmodium Species from Mangaluru, Southwest India

Malaria is a global threat and a never-ending battle without appropriate identification and differentiation of the parasite species. This work compared the diagnostic methods including the thick film microscopy technique, quantitative buffy coat, and polymerase chain reaction. The inaccuracy of species determination by microscopy and the consequent treatment regime underlines the necessity to upgrade routine diagnostic methods with molecular techniques. In the study, 436 samples were collected; venous blood was processed for the quantitative buffy coat technique followed by classical Giemsa staining of thin and thick smears and nested Polymerase Chain Reaction (nPCR) for the genus-specific region of Plasmodium targeting 18S rDNA followed by species-specific identification. Of 436 samples screened for malaria, results in PCR showed 78.7% (100/127) to be P. vivax, 4.8% (6/127) as P. falciparum and 16.5% (21/127) to be mixed infection (P. vivax + P. falciparum). The prevalence of malaria was 0.29, and there was good concordance between the methods for detecting Plasmodium (Kappa:0.77). In our investigation, nested PCR and TFM exhibited a sensitivity of 97.7% and a specificity of 100% for malaria detection compared to QBC. Clinical parameters- thrombocytopenia and anemia, were compared in this study. A positive association was observed between thrombocytopenia and malaria (p<0.05), but the association between anemia and malaria infection remains unclear. Primer cross-reactions were also observed in the primer sequence of P. ovale and P. knowlesi, but sequencing confirmed it as P. vivax and the study of phylogeny paved a new way in analyzing the relatedness of the sequences.

A trial study on a mixture of Acridine and Giemsa stains and modified Quantitative Buffy Coat for detection of malaria parasites

Microscopy after Geimsa staining remains the gold standard technique for detection of malaria parasites while Acridine is a common fluorescent stain that enhances the visibility of parasites. Combination of both stains could further enhance the performance of microscopy technique. The aim of this study was to investigate the efficacy of staining with mixture of Acridine and Giemsa and modified Quantitative Buffy Coat technique in detection of plasmodium parasites. Five hundred and seventy (570) volunteered students attending the Kwara State University Health Center Malete with history malaria fever and tested positive for malaria parasite were re-examined with the two methods being investigated. Two millilitres of the blood sample from each subject was collected into EDTA container and mixed properly. Thick smears were made in duplicate from each sample and properly labeled, stained by Giemsa and Acridine-Giemsa techniques and examined under x100 objective of the microscope while the remaining blood samples were also tested by Modified QBC. The study assessed the Sensitivity, Specificity, Positive Predictive Value and Negative Predictive Value of both techniques with respect to Giemsa microscopy as a gold standard. Findings from the investigation showed that modified QBC and Acredine-Giemsa techniques recorded sensitivity of 100% and 99.7% respectively. Concentrated Acridine-Giemsa stain recorded higher specificity (90.7%) than modified QBC with 82.5% using Geimsa staining as a gold standard. The Positive Predictive Value (PPV) for Modified QBC and Acredine-Giemsa techniques were 96.8% and 96.5% respectively whereas the Negative Predictive Value (NPV) for both methods were 100% and 98.7% respectively. In conclusion, the finding depicts a keen competition between Acridine-Giemsa staining method and Modified QBC technique for detection of malaria parasites and there was no significant difference in the efficacy of both methods with respect to detection of malaria parasite.© 2019 International Formulae Group. All rights reservedKeywords: Acridine-Giemsa, Modified QBC, Efficacy, Microscopy, Gold standard

Evaluation of Two Transfusion Transmitted Malaria Screening Methods at Kenyatta National Hospital, Kenya

Malaria is a disease responsible for morbidity and mortality in Sub-Saharan Africa. The primary mode of transmission is through the bite of an infected female Anopheles mosquito. There is transfusion transmitted malaria following blood transfusion in medical facilities. Specific objectives: This study was designed to determine the prevalence of malaria parasite among voluntary blood donors and to evaluate the performance characteristics of Quantitative buffy coat and Rapid diagnostic technique using CareStart TM malaria cassette from AccessBio manufacture were compared. Microscopy was used as the gold standard. Design: Cross sectional descriptive study. Subjects: Voluntary blood donors. Results: A total of 155 blood donors were recruited in this study and blood samples were screened for malaria parasites. The prevalence of malaria parasite using microscopy, rapid diagnostic test and quantitative buffy coat was 5/155 (3.2%), 8/155 (5.2 %) and 6/155 (3.9 %). The sensitivity of rapid diagnostic test and quantitative buffy coat was 80% and 100% while the specificity was 99% and 97%. The positive predictive values for rapid diagnostic test and quantitative buffy coat were 50 % and 83% while the negative predictive values were 99% and 100 %. Conclusion: This study concluded that some blood donors have malaria infection since prevalence of malaria parasite in blood donors was 3.2 %. Quantitative buffy coat technique was more sensitive and with higher specificity. Recommendations: This study recommends that blood and its products for transfusion should be screened for malaria parasites. The quantitative buffy coat technique should be used for malaria diagnosis.

‘In-house’ quantitative buffy coat technique in the diagnosis of malarial parasites: a cost-effective method

Background Microscopic examination of blood films is accepted as the universal ‘gold-standard’ for the diagnosis of malaria. However, low parasitemia accounting for false-negative results and time consumption for declaring results are limiting factors for smear microscopy. The quantitative buffy coat (QBC) technique is a more rapid test and can detect malarial parasites (MP) in the condition of low parasitemia. Aim The aim of this study was to show the usefulness of an ‘in-house’ QBC method in the diagnosis of MP. Patients and methods One hundred positive smear samples of patients diagnosed with malaria were subjected to analysis by both the ‘in-house’ QBC and the commercial QBC technique. Two hundred samples negative for malaria on microscopic smear examination were also tested using the ‘in-house’ QBC method. Results The 100 smear positive samples tested positive for MP with both commercial QBC testing and the ‘in-house’ QBC technique. Of the 200 samples that tested negative on smear examination, 16 were positive for malaria on testing with the ‘in-house’ QBC technique. Conclusion The described ‘in-house’ QBC technique provides an additional advantage of being cost-effective in comparison with the available commercial QBC kits.

A preliminary comparative report of quantitative buffy coat and modified quantitative buffy coat with peripheral blood smear in malaria diagnosis

The quantitative buffy coat (QBC) technique is a method of diagnosing malarial parasites based on micro-centrifugation, fluorescence, and density gradient of infected red blood cells. The aim of the present study was to modify the QBC technique in order to reduce the cost per test of malaria diagnosis. This was achieved by introducing some modifications to routine QBC wherein REMI centrifuge (cost Rs 19 000/–) and ultra-violet microscope (Rs 115 000) were used instead of parafuge (Rs 108 000) and paralens (Rs 293 625/–). With the above modification, the cost per test for laboratories dealing with high patient load was reduced by 13%, whereas for smaller laboratories with low patient load, the cost per test was reduced by 48%. This is a significant difference in cost. The results of the modified QBC method were compared with the current diagnostic methods: peripheral blood smear (PBS) and routine QBC. Blood samples collected from 96 patients were subjected to the above tests. Considering PBS as the gold standard, routine QBC showed 91% sensitivity and 96% specificity for Plasmodium vivax- and 91% sensitivity and 94% specificity for Plasmodium falciparum-infected patients. It was seen that the modified QBC technique had 91% sensitivity and 98% specificity for P. vivax and 91% sensitivity and 96% specificity for P. falciparum. It was concluded that modification of the QBC technique renders it cheaper without compromising the specificity and sensitivity of the method.

Comparison of Quantitative Buffy Coat technique (QBC) with Giemsa-stained thick film (GTF) for diagnosis of malaria

The renewed interest in the use of fluorescent microscopy for malaria diagnosis prompted the comparison of Quantitative Buffy Coat technique (QBC) with the old standard Giemsa-stained Thick blood Film (GTF) in Ikeja General Hospital, now Lagos State University Teaching Hospital, in Lagos. Blood samples were collected from 353 patients, each examined with the QBC and GTF techniques. Of these, 68 were positive with GTF, 70 with QBC giving a positive rate of 19.3% and 19.8% respectively. The malaria positive rate was calculated as 19.3% using GTF as the standard. In general, females recorded higher percentages (58.6% and 54.4%) than males (41.4% and 45.6%) among those positive with QBC and GTF respectively. The overall sensitivity rate for QBC was 55.9% and the specificity was 88.8%. The positive and negative predictive values of QBC compared to GTF were 54.3%, 89.4% respectively while the concordance of the two techniques was 82.4%. These values were lower than those reported for QBC in previous studies. The sensitivity of QBC reduced further (33.3%) with samples having low parasite density (< 1000 parasites/ul). QBC test was not able to accurately differentiate between different Plasmodium species but with the GTF, 86.7% of the infected individuals had Plasmodium falciparum, 7.5% had P. malariae and only 5.9% had mixed infections. In spite of the speed and simplicity of QBC technique, it cannot be considered an acceptable alternative to GTF under routine clinical laboratory situation. However, its speed and ease of use make it an important new tool for the diagnosis of malaria.

Comparison Between Thick Blood Films, Quantitative Buffy Coat Technique and the ParaSight-F Dipstick in the Diagnosis of Malarial Infections

Three different laboratory methods for diagnosing malarial in- fections, especially with Plasmodium falciparum, were evaluated in this study using thirty infected samples (22 samples contained Plasmodium falciparum, 8 samples contained P. vivax.). These methods included stained thick blood films, Quantitative Buffy Coat system, and Dipstrip (ParaSight-F) im- munoassay. Comparison among the three methods was done regarding the specificity to detect malaria parasites, the time required to perform the ex- amination, and the advantages and disadvantages of each method. ParaSight- F gave cross reactions with samples that contained high levels of rheumatoid factor (R.F.) and did not detect P. vivax. Quantitative Buffy Coat technique did not require technical skills but needed a long time to perform and species identification was very difficult. Thick blood films needed a longer time to be examined as well as special skills and experience, but it is still the method of choice since it can be applied in any laboratory or in the field with minimum equipments.

Short report: Diagnosis of tick-borne relapsing fever by the quantitative buffy coat fluorescence method.

The quantitative buffy coat (QBC) parasite detection method is a sensitive and specific tool for the diagnosis of malaria parasites. It is also useful for the diagnoses of other hemoparasites, including Trypanosoma, Babesia, and Leptospira. We report a case of relapsing fever diagnosed by this technique in a short-term traveler from Senegal. The diagnosis was confirmed by the standard Giemsa hemoscopy and by the identification of significant titers of antibodies to Borrelia spp. of tick-borne relapsing fevers by specific immunofluorescence and Western blot tests. The QBC technique seems to be useful in the diagnosis of tick-borne relapsing fever in blood samples and should be included in the management of fever in the traveler returning from tropical regions.

Comparative evaluation of four techniques for the diagnosis of Plasmodium falciparum infections

Four diagnostic techniques for Plasmodium falciparum infection were evaluated against serial parasite dilutions and on identical field samples. These were (i) Giemsa-stained thick blood films (GTF), (ii) acridine orange-stained thick (AOTF) and thin (AOTnF) blood films, (iii) the quantitative buffy coat technique (QBC ®); and (iv) the ParaSight TM-F dipstick test (PS). PS had a consistently higher sensitivity and speed, was easiest to learn, and required no laboratory facility. The 100% sensitivity cut-off points against known parasite densities (per mm 3) were: PS, 30; GTF, 84; QBC ®, 84; AOTnF, 84; AOTF, 149. In the field study, test sensitivities compared with examination of 800 microscope fields of a Giemsa-stained thin blood film were PS, 96·6%; AOTF, 93·1%; GTF, 91·4%; QBC ®, 89·7%; AOTnF, 82·8%. In the dilution study, one false positive result was recorded with QBC ®; in the field study there was one false positive each with PS, AOTnF and AOTF. When a newly trained microscopist examined samples of the parasite dilutions, the 100% sensitivity cut-off points were AOTF, 84; GTF, 140; QBC ®, 390. Total handling time was shortest with PS regardless of whether samples were processed individually or in batches of 10 or 100. The ParaSight TM-F test is recommended as the diagnostic tool for the future.

Isolation of Trypanosoma brucei gambiense from northern Uganda: evaluation of the kit for in vitro isolation (KIVI) in an epidemic focus

867 individuals from 3 sites near the town of Adjumani in the East Moyo region of north-west Uganda were investigated clinically and serologically for evidence of current trypanosome infections. Blood samples were taken from 94 persons with a positive card agglutination test for trypanosomiasis ( catt) and clinical suspects and inoculated into the kit for in vitro isolation of Trypanosoma brucei gambiense ( kivi). Amongst this group, 30 parasitaemic individuals were identified by microhaematocrit centrifugation and the quantitative buffy coat technique (QBC ®). Only 80% of these isolates, and one isolate from an aparasitaemic individual, grew in culture. The success or failure of cultures from parasitaemic patients was unrelated to the size of the trypanosome inoculum. The implications of these results and possible reasons for the failure of kivi are discussed.

THE LABORATORY DIAGNOSIS OF MALARIA.

Malaria particularly falciparum malaria is a major public health problem in India. Its correct and early diagnosis is very important for prompt treatment as a preventive and control measure. Microscopy is the traditional method for laboratory diagnosis of malaria, which is used widely. However, it is time consuming, needs expertism and the detection limit is 10-20 parasites/ml blood in thick film and 100 parasites/ml blood in thin film. Quantitative buffy coat technique (QBC) is highly sensitive method but expensive equipment is needed for this test. The serological methods involving antibody detection give information regarding exposure to malaria but do not differentiate between present and past infections. Genetic probes and PCR are highly sensitive methods but require expensive equipments. Parasite antigen detection tests are useful in field and PHC level for rapid diagnosis of P falciparum malaria.LDH based test for diagnosing malaria is sensitive but can not differentiate between species. For monitoring of drug resistance or follow-up of patients, methods which can quantify parasitaemia are needed. Simply microscopy is the best solution at present.