|Year : 2019 | Volume
| Issue : 2 | Page : 145-149
Utility of sunflower agar for laboratory detection of Cryptococcus neoformans
Sabitu Muhammad Zainu1, M Ayanbimpe Grace2, Yahya Mohammed3, BA Sani4, Aliyu Maryam Kangiwa1
1 Department of Medical Microbiology, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria
2 Department of Medical Microbiology, College of Medicine, University of Jos, Jos, Nigeria
3 Department of Medical Microbiology and Parasitology, Faculty of Basic Clinical Sciences, College of Health Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria
4 Department of Haematology and Blood Transfusion, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria
|Date of Submission||19-Aug-2019|
|Date of Decision||26-Sep-2019|
|Date of Acceptance||16-Oct-2019|
|Date of Web Publication||6-Dec-2019|
Dr. Sabitu Muhammad Zainu
Department of Medical Microbiology, Usmanu Danfodiyo University Teaching Hospital, Sokoto
Source of Support: None, Conflict of Interest: None
Background: Early diagnosis and management of cryptococcal meningitis is associated with good prognosis and long-term survival. Culture methods have been found to be promising and definitive in the diagnosis of many infectious diseases. We look at the sunflower agar (SFA) cultural method for detection Cryptococcus neoformans, the causative agent of cryptococcal meningitis. Materials and Methods: This is a descriptive cross-sectional study carried out at Jos University Teaching Hospital, Jos Nigeria, to find out the performance of SFA for the detection of C. neoformans. Cerebrospinal fluids (CSFs) of the study subjects were collected, subjected to Indian ink microscopy, inoculated on Sabouraud dextrose agar (SDA) and SFA for identification of C. neoformans. Results: A total of 90 CSF samples were analyzed for the identification of C. neoformans. SFA and SDA were able to confirm 8 (50%) and 7 (43%) of the 16 capsulated yeast cells detected by Indian ink microscopy as C. neoformans. Both media were found to have similar sensitivity (100%), specificity (91.3%), positive predictive value (80%), and negative predictive value (100%) in comparison with Indian ink microscopy. In terms of turnaround time, 6 isolates were identified within an average of 48 h (P = 0.017) by SFA, while SDA detects 2 isolates (P = 0.111) at the stipulated period. Conclusion: SFA can be a good routine conventional culture media for laboratory detection of C. neoformans.
Keywords: Cryptococcus neoformans, Indian ink, meningitis, meningoencephalitis, Sabouraud dextrose agar, sunflower agar
|How to cite this article:|
Zainu SM, Grace M A, Mohammed Y, Sani B A, Kangiwa AM. Utility of sunflower agar for laboratory detection of Cryptococcus neoformans. Ann Trop Pathol 2019;10:145-9
|How to cite this URL:|
Zainu SM, Grace M A, Mohammed Y, Sani B A, Kangiwa AM. Utility of sunflower agar for laboratory detection of Cryptococcus neoformans. Ann Trop Pathol [serial online] 2019 [cited 2020 Feb 26];10:145-9. Available from: http://www.atpjournal.org/text.asp?2019/10/2/145/272426
| Introduction|| |
Cryptococcus neoformans (C. neoformans) is found in the environment; it is abundant in the soil growing as a saprophyte. There was strong evidence that C. neoformans var. grubii evolved from Africa. It is commonly associated with pigeon and captive birds' droppings that provide appropriate conditions for its growth.,Cryptococcus species were also isolated from vegetables and fruits, house dust, air conditioners, air, and sawdust., The main route of transmission to human is inhalation from the environmental source and penetration through skin. Reactivation of a latent cryptococcal infection is the most common cause of opportunistic fungal meningitis, a life-threatening fungal infection in AIDS patients.,
Pulmonary infection is usually the first primary cryptococcal infection with possible pleural effusion,, from the lung it can spread to infect any organ. In immunocompetent host, pulmonary cryptococcosis is usually asymptomatic or associated with unusual presentation. The common species implicated with infection in immunocompetent individual is C. neoformans var. gattii which can present in a disseminated high mortality form. The most common presenting symptoms associated with pulmonary infections include cough, chest pain, sputum production, weight loss, fever, and hemoptysis. Rarely, it is associated with dyspnea, night sweats, and obstruction of the superior vena cava. Significant number of cases are associated with signs and symptoms of subacute meningitis or meningoencephalitis. Cryptococcal meningoencephalitis is the most common manifestation of cryptococcosis in immunocompromised individuals.,,,
C. neoformans infections cause an estimated one million cases of cryptococcal meningitis per year among people with HIV/AIDS and account for 625,000 deaths globally., The highest burden of disease was reported in sub-Saharan Africa with an estimated mortality of 50% to 70%.,, This high mortality in Sub-saharan Africa is linked to delay in presentation and late diagnosis which make early treatment difficult. Any approach leading to early diagnosis to improve early treatment of cryptococcal disease and its complications can reduce the associated high mortality. Prognosis is usually good when treatment start at early stage of the disease.
The laboratory detection methods for the identification of Cryptococcus species (spp.) include; microscopic detection of its polysaccharide capsular yeast cell in cerebrospinal fluid (CSF), isolation in culture, histopathology or by rapid biochemical identification systems or using rapid lateral flow assay for detection of cryptococcal antigen in serum and CSF., India ink preparations, alcian blue, and mucicarmine stains are used for microscopic identification of the capsulated Cryptococcus spp.,, The importance of using chromogenic like special media for the diagnosis of cryptococcal meningitis cannot be overemphasized. Various plants seed has been tried for that purpose, we chose sunflower seed to evaluate its performance in comparison with conventional Sabouraud dextrose agar (SDA) because of its availability in our environment and the simplicity of its preparation.
Helianthus annuus (sunflower) grows best in fertile, moist, well-drained soil with heavy mulch. It contains caffeic acid, a substrate for the production of melanin that is utilize by Cryptococcus spp. It is commercially being used as ornamental plants, production of butter (sunflower butter), food for birds, cooking oil (polyunsaturated oil), and medical ointments and dyes.
| Materials And Methods|| |
The study was conducted at the Jos University Teaching Hospital (JUTH). CSF samples collected from patients with suspected meningitis were used in this research. The laboratory procedures were conducted at the microbiology laboratory of JUTH, Jos, Plateau State, Nigeria.
This is a descriptive cross-sectional study.
Ninety CSF samples from suspected cases of meningitis were collected based on the following.
Subject selection criteria
Immunocompromised patients with symptoms suggestive of meningitis and immunocompetent CSF samples with a negative bacterial culture. CSF samples with established laboratory diagnosis of meningitis were excluded.
Preparation of simplified sunflower agar
Fifty grams of sunflower seed [Figure 1] was pulverized (crush to powder) in a domestic blender and boiled in 1 L of distilled water for 30 min. The seed extract obtained was allowed to be cooled and filtered through 5 layers of gauze and then 1 g glucose and 15 g agar powder (HIMEDIA-RM201) were added. Agar Powder purified (HIMEDIA RM201) is an extensively purified by exhaustively extracting Agar with water and organic solvents to remove all nitrogenous compounds, inorganic salts and vitamins. It has low calcium and magnesium levels and is compatible with all culture media. The pH was adjusted to 5.6 using hydrochloric acid and NaOH, and the volume made up to 1 L and autoclaved at 120°C for 15 min. The prepared medium was dispensed in sterile Petri dish More Detailses and slant in tubes. The prepared media was stored at a refrigerator temperature of 4°C–8°C.
The media maintained its performance for up to 8 weeks at the refrigerator temperature.
Preparation of Sabouraud dextrose agar
Commercially dehydrated SDA was purchased (HIMEDIA) and prepared according to the manufacturer guidelines. The composition was formulated, adjusted, standardized to suit performance parameters. Ingredients were dextrose 40.000 g/l, mycological peptone 10.000 g/l, agar 15.000 g/l with final pH at 25°C of 5.6. Sixty-five grams of the dehydrated powder was suspended in 1000 ml of distilled water, heated to boiling until the medium dissolved completely. It was then sterilized by autoclaving at 15 lbs pressure (121°C) for 15 min. The prepared medium was dispensed in Sterile Bijou bottles in slants and store at refrigerator temperature.
Indian Ink microscopy
Using sterile Pasteur pipette, a drop of Indian ink was placed at the center of clean, dried, grease-free, and microscopic glass slide. A drop of CSF was placed on the drop of the Indian ink. This was gently mixed using a small clean wooden applicator stick and covered with new clean coverslip. The wet mount preparation was then viewed at × 10 and × 40 for the identification of capsular yeast cells that gives presumptive diagnosis of C. neoformans as the causative agent of the disease. This procedure was also repeated on the in-house generated Cryptococcus species as a positive control.
Culture of cerebrospinal fluid on Sabouraud dextrose agar and sunflower agar
The collected CSF was kept in an incubator for 2 h and was then carefully decanted leaving lowest portion of about 0.5 ml (sediment), 0.5 ml CSF sediment was inoculated into each of SDA and sunflower agar (SFA) slant. In-house generated Cryptococcus species were also inoculated as positive control. All were incubated at 37°C for 24–72 h. The positive growth showed cream-colored smooth, mucoid yeast-like colonies on SDA. Yeast growth with characteristics brown color effect (BCE) on SFA gives preliminary identification of C. neoformans.
Lactophenol cotton blue wet mount
All growth that show characteristics yeast colonial morphology on culture were subjected to lactophenol cotton blue (LPCB) wet mount. Yeast cell with characteristics capsule gives preliminary identification of Cryptococcus species.
All growth that show characteristics yeast colonial grow pattern were inoculated on urea agar in small bijou bottle. This was incubated at 37°C for 48 h. The cultures that show characteristic pinkish color changes were considered urease positive.
Interpretation of results
The cultured specimen that show characteristic yeast colonial morphology, BCE on SFA, yeast cell morphology on LPCB wet mount, and urease positive were identified as Cryptococcus species in this study.
The data were analyzed using Epi Infotm7, 2013 and ISO Wizard statistical software. In all, critical P < 0.05 was regarded as statistically significant. Data were presented in tables.
Ethical approval was obtained from Ethical Committee of the JUTH.
| Results|| |
A total of 90 CSF samples were analyzed using Indian ink microscopy, SFA, and SDA cultures. All the results were ready within 72 h. A characteristic dark yeast cells surrounded by halo [Figure 2] were observed in 16 (17.78%) of the 90 CSF samples analyzed by Indian ink microscopy. A characteristic whitish mucoid like growths [Figure 3] were observed in 11 (12.22%) on SDA. Growth with BCE [Figure 4] was observed in 8 (8.8.00%) of cultured samples on SFA. In terms of urea hydrolysis [Figure 5], 7.7.00% and 8.800% of SDA and SFA were positive, respectively. A yeast-like morphological appearance was observed in all growths on both SDA and SFA by LPCB wet mount analysis. The rate of detection C. neoformans before urea hydrolysis by Indian ink, SDA, and SFA was found to be 16 (17.78%), 11 (12.22%), and 10 (11.11%), respectively [Table 1]. Urea hydrolysis results [Table 2] showed that 7 (7.78%) were positive for SDA and 8 (8.89%) positive for SFA.
|Figure 4: Culture growth on sunflower agar (sunflower agar) (a): Growth with no brown colour effect. (b) Growth with brown colour effect|
Click here to view
|Figure 5: Urea utilization reaction; Arrow pointing positive pinkish urea hydrolysis reaction|
Click here to view
|Table 1: Rate of isolation of Cryptococcus neoformans on Indian ink microscopy Sabouraud dextrose agar and sunflower agar|
Click here to view
|Table 2: Rate of urea hydrolysis from growth on Sabouraud dextrose agar and sunflower agar|
Click here to view
The turnaround time to the final presumptive identification of C. neoformans by SDA and SFA were also compared [Table 3]. After 24 h incubation, two results were ready for SFA and none for SDA, growth was also observed in 4 SFA and 3 SDA culture, respectively, after 48 hours' incubation. After 72 h, 1 culture was positive for SFA and 5 for SDA. In total, SFA and SDA were able to detect (confirmed) 6 and 1 isolates at 48 h, respectively.
|Table 3: Comparison of turnaround time between Sabouraud dextrose agar and sunflower agar to presumptive identification of Cryptococcus neoformans (n=8)|
Click here to view
In this study, growth on SFA with BCE and evidence of urea hydrolysis on SDA growth give presumptive identification of C. neoformans for the respective media. Indian ink microscopy, SDA, and SFA cultures were all 100% for both sensitivity and negative predictive value (NPV). In terms of specificity both SDA SFA was found to have a specificity of 91.3% with excellent positive predictive value (PPV) of 80% [Table 4].
|Table 4: Comparison of performance between Sabouraud dextrose agar and sunflower agar|
Click here to view
| Discussion|| |
High burden of cryptococcal meningitis among HIV-infected individual warrant a need of simple laboratory procedure for rapid diagnosis with high degree of specificity and accuracy. Indian ink microscopy is the common laboratory procedure employed for the laboratory diagnosis of cryptococcal meningitis. It is associated with high false positivity due to presence of leukocytes, myelin globules, fat droplets, tissue cells in the CSF, deteriorated reagent and when few organisms <103 colony-forming unit. In terms of culture, SDA culture method is the common cultural methods employed, but it requires further biochemical analysis for presumptive identification. Melanin production has been associated with virulence of C. neoformans in a number of studies. Media containing substrate (caffeic acid) for the production of melanin were recently being a focus of research for the laboratory isolation C. neoformans var. neoformans. Various plant seed known to contain caffeic acid were tried, these includes sunflower seed, mustard seed, tobacco seed, birdseed, and henna seed. Sunflower seed agar was found to be a simple and inexpensive tool for the presumptive identification of C. neoformans in clinical microbiology laboratories. SFA is simple to prepare as shown in this study, using readily available sunflower seed and commercially available agar powder. We obtained the seed at no cost from bushes around where the sunflower grow. The prepared media were stored at refrigerator temperature, and the performance was maintained for up to 8 weeks. It also has an excellent capacity for the isolation of C. neoformans. Its sensitivity, specificity, PPV, NPV, and degree of accuracy is as good as that of SDA as observed in this research which agreed with similar study conducted by Ruchi et al. in India. Its earlier statistically significant turnaround time (P < 0.017) as compared to that of SDA (P < 0.111) helps in earlier diagnosis and warrant possibility early initiation of treatment. In this study, SFA was found to be associated with a high degree of accuracy (91.3%) in establishing the diagnosis of cryptococcal meningitis; its high specificity limits the possibility of having high false-positive cases.
| Conclusion|| |
Readily available sunflower seed and simplicity in preparing the medium can make SFA a good chromogenic culture method for the laboratory diagnosis of cryptococcal infections in a poor resource setting.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Cabral Passoni LF. Wood, animals and human beings as reservoirs for human Cryptococcus neoformans
infection. Rev Iberoam Micol 1999;16:77-81.
Anastasia PL, Ignazio C, Jenny R, Rameshwari T, Nelesh PG, Thomas GM. Evidence that the human pathogenic fungus Cryptococcus neoformans
vr. Grubii may have evolved in Africa. PLoS One 2011;6:1-13.
Irokanulo EO, Makinde AA, Akuesgi CO, Ekwonu M. Cryptococcus neoformans
isolated from droppings of captive birds in Nigeria. J Wildl Dis 1997;33:343-5.
Bicanic T, Harrison TS. Cryptococcal meningitis. Br Med Bull 2004;72:99-118.
Mahendra P, Sihin T, Pratibha D. Cryptococcosis: An enigmatic mycosis of humans and animals. J Environ Occup Sci 2014;3:53-60.
French N, Gray K, Watera C, Nakiyingi J, Lugada E, Moore M, et al.
Cryptococcal infection in a cohort of HIV-1-infected Ugandan adults. AIDS 2002;16:1031-8.
Antinori S. New insights into HIV/AIDS-associated cryptococcosis. ISRN AIDS 2013;2013:471363.
Chang MR, Paniago AM, Silva MM, Lazéra MS, Wanke B. Prostatic cryptococcosis a case report. J Venom Anim Toxins Incl Trop Dis 2008;14:378-85.
Carla JW, Wendy G, Sarah EH, Floyd W, Shawn RL, Julie RH, et al
. Fatal disseminated Cryptococcus gattii
infection in new Mexico. PLoS One 2011;6:1-8.
Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 2009;23:525-30.
Kanchan M, Satish P, Ravikumar N, Ramanath M. Prevalence of cryptococcal meningitis immunocompetent and immunocompromised individuals in Bellary, South India a prospective study. J Clin Diagn Res 2012;6:388-92.
Gomerep SS, Idoko JA, Ladep NG, Ugoya SO, Obaseki D, Agbaji OA, et al.
Frequency of cryptococcal meningitis in HIV-1 infected patients in North Central Nigeria. Niger J Med 2010;19:395-9.
World Health Organization. Rapid Advice. Diagnosis, Prevention and Management of Cryptococcal Disease in HIV-Infected Adults, Adolescents and Children; 2011. Available: http://www.who.int/hiv
. [Last accessed on 2014 May 17].
McTaggart L, Richardson SE, Seah C, Hoang L, Fothergill A, Zhang SX. Rapid identification of Cryptococcus neoformans
, C. neoformans
, and C. gattii
by use of rapid biochemical tests, differential media, and DNA sequencing. J Clin Microbiol 2011;49:2522-7.
Lentz DL, Pohl MD, Alvarado JL, Tarighat S, Bye R. Sunflower (Helianthus annuus
L.) as a pre-columbian domesticate in Mexico. Proc Natl Acad Sci U S A 2008;105:6232-7.
Ruchi K, Sachin CD, Santosh S. Comparison of different media for the pigment production of Cryptococcus neoformans
. J Clin Diagn Res 2011;5:1187-9.
Abassi M, Boulware DR, Rhein J. Cryptococcal meningitis: Diagnosis and management update. Curr Trop Med Rep 2015;2:90-9.
Williamson PR. Laccase and melanin in the pathogenesis of Cryptococcus neoformans
. Front Biosci 1997;2:e99-107.
Nandhakumar B, Menon T, Kumar CP. A new henna-based medium for the differentiation of Cryptococcus neoformans
. J Med Microbiol 2007;56:568.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]