Histoplasmosis is a fungal infection caused by Histoplasma capsulatum (H. capsulatum ) a dimorphic fungus that, can affect both humans and animals [1-3]. Most Histoplasma infections are subclinical, however, occasionally disease occurs linked in to the host immune response [4]. The disease is the most common pulmonary mycosis in North America and Central America, but the organism is known to exist in many areas around the world [5-9]. Histoplasmosis usually presents as in immune-compromised individuals with serious underlying disease, including AIDS [10,11]. The occurrence of histoplasmosis in Asia has been recently reviewed with cases of histoplasmosis from India, Malaysia, Singapore, Indonesia, Thailand, South Vietnam and Japan, but the autochthonous nature of the cases reported has not been established unequivocally [8,12,13]. Recently, H. capsulatum was recovered from environmental sources contaminated with bat guano and chicken droppings in Thailand [14]. In Vietnam, two histoplasmosis cases were reported from Southern Vietnam, however, the endemicity of the disease has not been studied. Histoplasmosis is difficult to distinguish from diseases with similar clinical features, such as tuberculosis and bacterial pneumonia. In the laboratory, diagnosis of histoplasmosis is also problematic because the culture-positive rate in patients is usually low, and several weeks are required for H. capsulatum to grow on solid media. In addition, H.capsulatum is a biosafety level 3 pathogen requiring specialist facilities for culturing. Serological methods are faster than culture, but can sometimes generate false positives via cross reaction with other fungal pathogens such as Coccidioidomycosis [3]. Moreover, in cases in which a histopathological diagnosis of histoplasmosis has been made, confusion can occur with other pathogens such as Cryptococcus spp ., or Penicillium marneffei , which can have similar pathologies. Molecular methods such as nested PCR and sequencing are alternatives for rapid detection of Histoplasma .

Here, we aimed to determine the prevalence of histoplasmosis among pulmonary infection patients in Hanoi, Vietnam using different diagnostic methods.

Sample collection and preparation for culture

Between August 2012 and January 2013, 206 serum and 158 bronchial washing samples were collected from pulmonary infection patients at Bach Mai and Military 103 hospitals in Hanoi. Criteria for enrolment of patients were at least one of the followings: i) abnormal chest shadow and clinical symptoms such as cough, sputum (haemptylis), or dyspnea; ii) negative mycobacterial examination (smear negative or culture negative). Each patient was asked to provide blood and bronchoalveolar lavage fluid (BALF) sample. Aliquots of the BALF were centrifuged and the supernatant was removed. Sterile PBS was added to the sediments, which were thoroughly mixed before being used to inoculate fungal cultures. Serum was obtained by centrifuging at 2000 g (or rpm) for 10 min at room temperature and was stored at -300°C until analysed.

ELISA

Serum samples were analysed using HistoplasmaDxSelect™ kit (USA) following the manufacturer’s instructions. ELISA results were compared with the reference cut-off OD value reading using an ELISA reader. An index value of >1.10 was used to indicate the presence of antibodies to Histoplasma . An index value of <0.90 indicated that antibodies to Histoplasma were not detected.

Nested PCR

DNA extraction from patient’s samples was performed using QIAamp® DNA mini kits in a 50 μl elution volume following the manufacturer’s instructions. Five microliters of extracted DNA was used for the nested PCR assay. Primers used in the assay to detect H. capsulatum specifically amplified a sequence of DNA coding for a specific portion of the H. capsulatum M antigen gene (Msp1F/Msp2R and Msp2F/Msp3R) (Table 1) [15].

Table 1: Histoplasma capsulatum PCR specific primers.

Sequencing

The second nested PCR amplicons were purified using the QIA quick PCR purification kit (Qiagen).The purified products were sequenced directly in both directions using a Big Dye Terminator v3.1 Cycle sequencing kit (Life Technologies) in an Applied Bio system 3130 genetic Analyser (Life Technologies). Nucleotide sequences for the 269 bp products were verified using the National Centre for Biotechnology Information, Basic Local Alignment Search Tool.

Culture

One hundred microliter aliquots of each PCR-positive samples were cultured onto Brain Heart Infusion agar (Merck) with 1% glucose. Plates were incubated at 28-30°C for up to 8 weeks. Colonies were identified by microscopic morphology.

Ethical review

The study was approved by the ethics committee of the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam (No. 01 IRB, dated January 6, 2012).

Histoplasmosis is a community acquired infection, most often presenting as pneumonia [1]. Although histoplasmosis is not a common cause of lung disease in the tropics, it is probably underdiagnosed due to the technical difficulties of microbiological diagnosis and the likelihood of confusion with tuberculosis. Histoplasmosis diagnosis is usually based on visualization of the fungus in organic fluids (sputum, blood and liquor) or tissues (histopathological evaluation) and by the culture of biological samples. The present study used HistoplasmaDxSelect™ ELISA kit, which utilizes inactivated purified Histoplasma antigen for the qualitative detection of antibodies to Histoplasma capsulatum var. capsulatum in human serum. In this study, a total of 206 serum and 158 bronchial lavage samples were collected from patients with acute or chronic pulmonary infections during 2012-2013 (Table 2). The acute pulmonary group (90/206) was hospitalized with typical symptoms as dry or nonproductive cough, fever, chest pain and fatigue. Distinct patterns were occasionally seen on a chest X-ray. The chronic pulmonary patients (116/206) were commonly in older men who had the clinical manifestations include fatigue, mild fever, night sweats, chronic cough, sputum production and weight loss. Of the 206 patient serum samples tested for antibody reactivity by ELISA, Histoplasma antibodies were detected in 13.1% (27/206) which is high compared with studies form Japan and Thailand [1,5]. Positive samples were not significantly linked to occupational group with 16/206 (7.8%) from farmers, 7/206 (3.4%) from workers and 4/206 (1.9%) from other groups, respectively (Table 3). Presence of Histoplasma antibodies was high in chronic pulmonary patients compared to the acute pulmonary group 15.5% (17/116) and 10% (9/90) respectively. Of the HIV-infected patients 42.8% (15/35) were identified positive with Histoplasma antibodies (Table 3). The higher prevalence of antibodies in farmers might be explained by their close contact to soil and material contaminated with bird/chicken dropping. In Thailand, Pirron et al. showed 90.6% of histoplasmosis patients were Acquired Immunodeficiency Syndrome (AIDS) [5]. However, those patients were positive with H. capsulatum by culture while our patients were cases that diagnosed by clinical signs. In addition, we found 8.74% of chronic pulmonary patients were seropositive, compared with 4.36% with acute pulmonary patients. This could be due to the development of anti-Histoplasma antibodies occurring two to six week’s post-exposure [6]. In acute case where Histoplasma infections is suspected, a second serum, sample should be collected 7-14 days later for testing to avoid false negative results [6]. The serological should be only used as a screening test for evaluation potential exposure to, or infection with, Histoplasma and confirmatory are required.

Table 2: Characteristics of patients included in the study.

Table 3: Comparison of ELISA results in the patients group.

Several reports suggested that PCRs might improve the accuracy of identification of H. capsulatum in clinical samples such as tissues, body fluids. In this study, we used a nested PCR with the specific primers for detection of H. capsulatum in both type of clinical samples. Detail of the primers were previously published but were modified by Ohno H et al. to increase the sensitivity and specificity of PCR allowing for detection of at least 10 fg H. capsulatum DNA [15]. In the present work, 9/206 BALF samples were positive with the nested PCR (Figure 1) and sequence analysis of these PCR products showed approximately 100% similarity to the H. capsulatum M antigen gene. Among these PCR positive samples, 3 of them correlated with a positive ELISA results. While this provided strong evidence that those patients were infected with H. capsulatum , isolation of H. capsulatum from PCRpositive samples by culture was negative after 8 weeks of the incubation period. This could be due to deterioration of the samples during long-term storage or that patients had undergone long-term antibiotic therapy. Therefore, knowledge of clinical syndromes and the patient travel history may lead the physicians to decide sending sample on time and avoid inappropriate treatment regime.

Figure 1: Nested PCR results in pulmonary infection patients.

Our results provide evidence that histoplasmosis should be considered in patients with pulmonary infections in Hanoi, Vietnam and that such infections may be under-reported. Physicians should therefore include histoplasmosis in the differential diagnosis of diseases thought to be tuberculosis or community-acquired pneumonia. In addition, diagnosis of histoplasmosis should include culture to allow validation of the non-culture diagnostic technique used in this study in the Vietnam setting. Adaptation and validation of the Histoplasma antigen tests for use in low resource setting could assist with recognition of patients with the infection, allowing early intervention with an appropriate treatment regime.

This work was supported by a grant-in-aid of Ministry of Health, Labour and Welfare, the Government of Japan (H23-Shinkoushitei- 020) and Japan International Cooperation Agency (JICA).