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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 116-120

Co-infection by dimorphic fungi in tuberculosis patients in Kenya


1 Department of Veterinary Pathology, Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Nairobi, Nairobi, Kenya
2 Department of Medical Services and Public Health, Ministry of Health Laikipia County, Nanyuki, Kenya
3 Department of Medical Laboratory Sciences, Ministry of Health Laikipia County, Nanyuki, Kenya

Date of Web Publication29-May-2020

Correspondence Address:
Joseph K Ngei. Kuria
Department of Veterinary Pathology, Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Nairobi, Nanyuki
Kenya
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_44_20

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  Abstract 


Background: Dimorphic fungi may cause infections and symptoms similar to tuberculosis (TB), in humans and animals. Such infections, individually or concurrently with TB, have been identified in cattle in Kenya, raising the possibility of infections in other animals, including humans. The study aimed to identify and quantify dimorphic fungi co-infection in persons with TB. Methods: Smear-positive sputum samples, 400, were obtained from TB clinics between October 2016 and November 2017. The samples were examined microscopically for yeast fungi, cultured for isolation of yeast, conversion to molds, and conversion from molds to yeasts. The isolates were characterized morphologically. Results: Blastopores, with morphological characteristics of Paracoccidiodes and Blastomyces, were observed in 37 smears of the sputum samples. Similar yeast cells were observed in smears of the sputum cultures. The yeast cultures were converted to molds on incubation at room temperature and back to yeasts on incubation at 37°C. Conclusion: Dimorphic fungi, morphologically identified as Paracoccidiodes and Blastomyces, concomitantly infect a proportion of TB patients in the study area. It is recommended that routine diagnosis for TB should consider infection or co-infection by dimorphic fungi for institution of appropriate treatment.

Keywords: Co-infection, dimorphic fungi, tuberculosis patients


How to cite this article:
Ngei. Kuria JK, Mogoi D, Gachuhi SG. Co-infection by dimorphic fungi in tuberculosis patients in Kenya. Int J Mycobacteriol 2020;9:116-20

How to cite this URL:
Ngei. Kuria JK, Mogoi D, Gachuhi SG. Co-infection by dimorphic fungi in tuberculosis patients in Kenya. Int J Mycobacteriol [serial online] 2020 [cited 2020 Aug 3];9:116-20. Available from: http://www.ijmyco.org/text.asp?2020/9/2/116/285230




  Introduction Top


Tuberculosis (TB) has re-emerged as an important public health problem worldwide, and Kenya is one of the thirty high-burden countries,[1] hence accurate diagnosis is important in the surveillance, treatment, design, and implementation of control programs. Agents other than mycobacteria, which include other bacterial genera, parasites, and fungi, may cause infections, with symptoms and gross lesions similar to TB.[2],[3] Dimorphic fungi, which include, Blastomyces dermatitidis, Paracoccidioides spp, Histoplasma capsulatum, Coccidioides immitis, and Telaromyces (Penicillium) marneffei, are such agents and cause chronic granulomatous pulmonary or disseminated infections resembling TB.[4],[5],[6] Spores of the mold form are infections and are found in soil, hay, and plants and usually affect farmers, gardeners, hunters, and agricultural workers, especially those that are immunocompromised.[7]B. dermatitidis and H. capsulatum have a worldwide distribution, but others (T. marneffei, Paracoccidioides brasiliensis and C. immitisis) are said to be geographically restricted, with Paracoccidioides being restricted to South and Central America[8],[9],[10] Outside Latin America, paracoccidioidomycosis is regarded as a disease of travelers, being observed in persons individuals who had previously visited or resided in the endemic region.[11],[12],[13] Infection occurs through inhalation of airborne fungal conidia and persons at risk are those involved in agriculture and cattle rising. Coffee and tobacco farming has been found to involve a higher risk of inhaling Paracoccidioides conidia present in soil[14]B. dermatitidis infections are reported mostly in humans and dogs in North America, but some isolated human cases have been reported in Africa, Israel, India, and Bangladesh.[15],[16] The mold spores survive in soil that contains organic debris and infects people engaged in activities that disrupt the soil such as collecting firewood or tearing down old buildings.[17],[18] The identification of Paracoccidioides and B. dermatitidis infections, separately or concurrent with TB in slaughter cattle in Kenya,[3] raised the possibility of infection in other animals, including humans. This study aimed to determine and estimate the prevalence of dimorphic fungi and TB co-infection in clinically presumed persons in Kenya. Findings from the study will strengthen TB diagnosis leading to effective management and control.


  Methods Top


Study site/setting

The study comprised sputum sample collection and laboratory analysis. Sputum samples were collected from two hospitals and one dispensary in Laikipia County, Kenya. In the health facilities, persons with TB symptoms are screened for the disease by examination of acid-fast stained sputum samples in designated clinics and those found smear positive are put on medication. Those with persistent symptoms, even though smear negative, are also put on medication. Sputum samples are destroyed by incineration soon after examination and recording of results. The County was selected because of the rural agricultural setting and a high prevalence of TB. Laboratory analysis was carried out at the Department of Veterinary Pathology and Microbiology, University of Nairobi, the Central TB Reference Laboratory, Ministry of Health.

Sample collection

Sampling was done between October 2016 and November 2017. Because of the high volume of available samples, only smear-positive ones were collected. There were no ethical considerations as samples collected were set for destruction and identity of the patients was not recorded. The samples were labeled, placed into a cooler-box, transported to the laboratory, and stored at −20°C till processing.

Laboratory analysis

Sputum samples were examined for acid-fast bacilli (AFB) and yeast cell and then cultured for fungal isolation. The isolates were stained, examined, and characterized morphologically. All manipulations were carried out in biosafety cabinet Level II. Samples were allowed to thaw at 4°C for a day before processing. The samples were transferred from the sputum pots into 50 ml sterile Falcon® tubes and labeled, and equal volumes of sodium hydroxide/N-acetyl-L-cysteine-sodium citrate (NAOH-NALC) solution were added to decontaminate. A previous investigation indicated that the yeast cells can withstand this treatment.[3] The tubes were then tightly closed, vortexed for 5 min, and left to stand for 10 min to allow aerosols to settle. Phosphate-buffered solution (PBS), pH 6.8, was added to the full mark of the tube to stop decontamination. The suspension was then centrifuged at 800 × gfor 30 min at 4°C. The supernatant was discarded into a 5% phenol solution, and the pellet was re-suspended in 3 ml PBS containing benzylpenicillin, 50 IU per ml, by vortexing. Smears were prepared with one drop of the suspension and Ziehl–Neelsen staining was carried out following the standard procedure. Stained slides were examined under oil immersion (×1000) for AFB and yeast fungi. One hundred microliters of the suspension was inoculated into yeast extract peptone dextrose broth yeast peptone dextrose (YPD, Oxoid) containing 10% defibrinated horse plasma (YPD horse plasma broth) and 50 μg/ml of chloramphenicol and gentamycin each. The cultures were incubated at 37°C for 2 weeks and Gram-stained smears were examined for yeast fungi.

Demonstration of dimorphism

Selected yeast fungi cultures were inoculated into YPD agar slants. Safety precautions were taken as follows: the rubber lining of universal bottle metal caps was removed and a needle hole was drilled though the center of the cap. The rubbers were replaced, and the media slants were prepared in the bottles. One hundred microliters of yeast cultures was inoculated onto the slants, which were then incubated, inside a cabinet, at room temperature and observed for 7 days. Formal saline was injected into the mold cultures through the needle holes and after 24 h, mold samples were stained with lactophenol cotton blue stain and examined. Conversion from mold to yeast form was demonstrated by inoculating mold cultures into YPD horse plasma broth and YPD horse plasma agar slants. YPD broth, 2 ml, was injected into the mold cultures and the slants agitated gently. One hundred microliters of the supernatant were then withdrawn with a syringe and inoculated into the media which were then incubated at 37°C for 4 weeks. Smears of the cultures were then stained by Gram stain method and examined.


  Results Top


Examination of sputum samples

A total of 400 sputum samples were collected. AFB were observed in all samples, while in 37 (9.2%), yeast cells were observed together with mycobacteria. The yeast cells comprised of three morphological types, designated as Types 1, 2, and 3. Type 1 were either nonacid fast (blue), partially acid fast (purple), or acid fast (red) blastopores with single buds formed on a wide base [Figure 1]a and [Figure 1]b, observed in nine (2.4%) of the 37 samples. Type 2 comprised none-acid fast cells bearing two buds or forming pseudomycelia [Figure 1]c observed in five smears (13.5%) of the samples, while Type 3 were none -acid fast cells, either single, bearing a single bud or in short chains [Figure 1]d observed in 22 (59.5%) of the samples. One smear (2.7%) contained both Type 2 and 3 cells.
Figure 1: Photomicrographs of yeast fungi in smears of sputum samples from tuberculosis patient in Kenya. (a and b) Blastomyces dermatitidis cells showing single buds on a broad base; (c and d) Paracoccidiodes in single cells (1) single buds, (2) double buds, (3) short chains, (4) and pseudomycelia (5). Acid-fast bacilli = acid fast bacilli (Ziehl–Neelsen × 1000)

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Sputum culture

Growth in broth cultures was obtained in 19 of the 37 samples. Yeast cells in Type 2 cultures were morphologically similar to those in sputum smears. Large, intensely Gram-positive cells bearing two buds, or a mass of interlinked cells forming pseudomycelia [Figure 2]a and [Figure 2]b, were observed. Type 3 cultures formed smaller Gram positive single cells, cells bearing single or multiple buds or short chains of cells [Figure 2]c.
Figure 2: Photomicrographs of yeast fungi cultured from sputum samples from a tuberculosis patient in Kenya, (a-c) yeast peptone dextrose broth sputum culture smears showing Paracoccidiodes double buds (1) pseudomycelia (2) and multiple buds (3); (d) yeast peptone dextrose broth mold culture smear showing Paracoccidiodes double “mickey mouse” buds. (Gram stain × 1000)

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Demonstration of dimorphism

Broth yeast cultures of Type 2 and 3 cells were inoculated into YPD agar slants and incubated at room which produced white mold colonies of about 5 mm diameter in about 7 days. With continued incubation, the molds formed radially folded, glabrous colonies, with a tan reverse pigmentation. Microscopically, the molds comprised of septate hyphae with scarce ovoid conidia at the tips of the hyphae.

Molds cultures inoculated into YPD broths produced yeast cells similar to those described for sputum cultures. The blastospores, however, tended to stain Gram-negative, and in case of Type 2, smaller and with no tendency to form pseudomycelia. Cells typically bearing two or multiple buds were observed [Figure 2]d and [Figure 3]a. Molds inoculated into YPD horse plasma agar slants produced colonies, which at 4 weeks were round, about 2 mm diameter, raised, rough, and with a granular consistency [Figure 3]b. Microscopically, culture smears stained by Gram stain method consisted of a mixture of hyphae and blastopores [Figure 3]c and [Figure 3]d. From the characteristic blastopores with thick walls and single buds formed on a wide base, and blastopores with two or multiple buds morphology, the yeasts were identified as B. dermatitidis and Paracoccidiodes spp., respectively.{Figure 2}
Figure 3: Photomicrographs of yeast fungi cultured from sputum samples from tuberculosis patient in Kenya, (a) yeast peptone dextrose broth mold culture smear showing Paracoccidiodes multiple buds arranged in “mariners wheel” pattern (Gram stain × 1000); (b) Paracoccidiodes colonies from mold culture on yeast peptone dextrose agar; (c and d) smears from Paracoccidiodes colonies showing mixture of budding cells and hyphae. (Gram stain × 1000)

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  Discussion Top


Routine diagnosis of TB is mainly based on the examination of stained sputum smears for the causative agent, whereupon positive cases are put on medication. Cases that are smearing negative but with persistent symptoms are presumed positive and similarly put on medication. Other infectious agents that may cause symptoms similar to TB are rarely considered or investigated. In this investigation, 9.2% of TB patients were found co-infected with dimorphic fungi identified as Paraccocidiodes and Blastomyces. Currently, there are five recognized genera of dimorphic fungi. Four of the genera have only one species and the fifth two. Each genus has a distinct morphology either in yeast form, mold form, or both. Paraccocidiodes yeast form has two characteristic cellular morphologies, considered diagnostic for this fungus: a large mother cell bearing narrow-necked multiple budding yeasts, resembling a “mariners wheel” or mother cells with only two large buds, resembling a “Mickey mouse” head.[13] The two morphological types were identified in this investigation. Two species in the genus, P. brasiliensis and P. lutzii, are now recognized.[19]

Paracoccidiodes infections have hitherto been considered restricted to desert lands of South and Central America, and cases reported outside the endemic region are considered imported, being observed in only in persons who have previously visited or resided in the region[11],[12],[13]. However, identification of infection in cattle in Kenya[3] indicated that native Paracoccidioides infections may be misdiagnosed. A prevalence of 7% co-infection was observed in this investigation. In the endemic regions of South and Central America, paracoccidioidomycosis has been found to occur concurrently with TB in 15%–20% and 28.4%, respectively, of patients with TB.[20],[21]

B. dermatitidis was identified on the basis of the typical blastospores with a thick wall and a single bud with a wide base.[22],[23] Due to the thick cell wall, some blastospores in sputum smears were found to stain acid fast. Majority of B. dermatitidis infections have been reported in humans and dogs in North America. In Africa, Blastomycosis has been reported as individual human cases in 16 counties including Zimbabwe,[24] Tunisia,[16]South Africa, Nigeria, Zambia, Congo, and Gambia[25] and in slaughter cattle in Kenya,[3] indicating that infections may be widespread in the continent. A prevalence of 2.3% was observed in this investigation.

Dimorphic fungi infection has serious implications in diagnosis because of the clinical and pathological similarities to TB. Reports from the Kenya National TB Program indicated that about 60% of case notification is composed of smear-negative TB.[26] Such cases may be inappropriately treated for TB on the basis of clinical signs, hence the need for specific diagnosis. Co-infection of dimorphic fungi with TB may enhance symptoms and further complicates treatment because individual pathogens have to be targeted, with the possible risks of drug interactions. Persons infected with Paracoccidiodes may also develop pulmonary fibrotic sequelae and reduced adrenal function.[20] This investigation has established that Paracoccidiodes and Blastomyces concomitantly infect a proportion of TB patients in the study area. The investigation documents the first native human paracoccidioidoomycosis, outside the presumed geographically restricted region, and the first human blastomycosis in the country. Routine diagnosis for TB in the region should therefore consider infection or co-infection by dimorphic fungi to ensure appropriate treatments and effective control programs. A wider study, which should include determination of the proportion of patients infected with dimorphic fungi only, correlation between infection and clinical disease, risk factors for infection, as well as molecular identification of Paracoccidiodes species, is recommended.


  Conclusion Top


This investigation has established that Paracoccidiodes and Blastomyces concomitantly infect a proportion of TB patients in the study area. The investigation documents the first native human paracoccidioidoomycosis, outside the presumed geographically restricted region, and the first human blastomycosis in the country. Routine diagnosis for TB in the region should therefore consider infection or co infection by dimorphic fungi to ensure appropriate treatments and effective control programs. A wider study, which should include determination of the proportion of patients infected with dimorphic fungi only, correlation between infection and clinical disease, risk factors for infection, as well as molecular identification of Paracoccidiodes species, is recommended.

Acknowledgments

The authors would like to thank the National Council for Science, Technology, and Innovation for partial funding of the study and the National TB Reference Laboratory for provision of facilities.

Financial support and sponsorship

Partial funding of the study was provided by the National Council for Science, Technology, and Innovation.

Conflicts of interest

There are no conflicts of interest.



 
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