key: cord-0831205-i58ebccz
authors: Erami, Mahzad; Raiesi, Omid; Momen-Heravi, Mansooreh; Getso, Muhammad Ibrahim; Fakhrehi, Mojtaba; Mehri, Narges; Yarahmadi, Mohammad; Amiri, Sasan; Raissi, Vahid; Hashemi, Seyed Jamal
title: Clinical impact of Candida respiratory tract colonization and acute lung infections in critically ill patients with COVID-19 pneumonia
date: 2022-04-09
journal: Microb Pathog
DOI: 10.1016/j.micpath.2022.105520
sha: 8cbec504d9a564bb708ff2043588ebfbca594b54
doc_id: 831205
cord_uid: i58ebccz

Coronavirus disease 2019 (COVID-19), which is attributable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been causing a worldwide health issue. Airways colonization by Candida spp. is prevalent among patients on automatic ventilation in intensive care units (ICUs). This research aimed to ascertain the risk factors and roles of Candida spp. respiratory tract colonization, and Candida lung infection during the progression of COVID-19 pneumonia in critically ill patients. In total, Candida spp. were recovered in 69 from 100 immunosuppressed patients with COVID-19. Bronchoscopy was used to collect the Bronchoalveolar lavage (BAL) specimens. For the identification of Candida spp. PCR sequencing was done using the ITS1 and ITS4 primers. The amplification of the HWP1 gene was conducted to identify the Candida albicans complex. The antifungal activities of fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin against Candida spp. were evaluated using the Clinical and Laboratory Standards Institute M60. In 63.77% of the patients, Candida respiratory colonization at D0 and D14 had no impact on the severity of COVID-19. In comparison to C. albicans strains, Candida respiratory disorder with C. glabrata had influenced the severity of COVID-19 for critically ill patients following adjustment for the risk factors of COVID-19 (P < 0.05). Amphotericin B and caspofungin showed superior activity against all Candida spp. All antifungal agents showed 100% sensitivity against the two C. africana strains. Our observation on patients who used automatic ventilation, respiratory colonization by Candida spp. was not seen to influence the infection or death caused by COVID-19. Amphotericin B and caspofungin showed superior activity against all Candida spp. and were recommended for the treatment regime of pulmonary candidiasis associated with COVID-19 infection. Although “Candida pneumonia” is rarely being reported in critically ill patients, Candida airway colonization mainly by Candida albicans is common especially among patients with diabetes, malignancies, and kidney disorders.

The novel coronavirus SARS-coV-2, which emerged in Wuhan in November 2019, has increasingly spread 64 causing a global pandemic that infected more than 494 million people, resulting in severe social and 65 economic ramifications, and claimed more than 6,183,000 lives by April 6, 2022 [1] . Coronavirus disease 66 2019 (COVID-19), which is attributable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-67

2), has been the cause of global health threats [2, 3] . Bacterial and fungal co-infections are among various 68 factors that play roles in morbidity and mortality in COVID-19 patients, particularly among those suffering 69 from acute respiratory distress syndrome (ARDS). Furthermore, the wide use of corticosteroids and the 70 irrational use antibiotics coupled with the tissue damage caused by SARS CoV-2, may facilitate invasion 71 by commensal yeast causing deep seated invasive fungal infections. Patients with severe COVID-19 are at 72 risk for healthcare-associated infections (HAIs), including Candida bloodstream infections. There have 73 been reports on increasing incidence of candidemia in critically ill COVID-19 cases. High mortality rate is 74 being reported among patients with COVID-19-associated candidemia (CAC). The mortality rate among 75 patients with CAC reaches up to 83% despite antifungal therapy. The above highlights the clinical 76 significance of severe COVID-19 that underscores the importance of rapid diagnosis and timely initiation 77 of antifungal treatment [4] [5] [6] [7] . Moreover, the undefined standard of pharmacological therapy for COVID-19, 78

including the invasive nature and multi-drug treatment methods, as well as some pathological oral 79 conditions can aggravate SARS-CoV-2, particularly in those patients with a immune-compromised system 80 or a long-term usage of pharmacotherapies that expose them to increased risk for developing mucosal 81 candidiasis [8] . Bronchial colonization by Candida spp. is prevalent among patients who use automatic 82 ventilation in the intensive care unit (ICU). Candida colonization has been found in approximately 30% of 83 people who used mechanical ventilation (MV) for longer than 48h and in 50% of those diagnosed with 84 According to the manufacturer's instructions, genomic DNA was extracted directly from BAL specimens 116 using a high-purity polymerase chain reaction (PCR) template purification package (Roche, Germany). 117

Briefly, 200 μl of specimens were mixed with 200 μl of binding buffer and 40 μl of proteinase K. The 118 mixture was incubated at 70 °C for 10 min followed by the addition of 100 μl of isopropanol. A high-purity 119 filter tube was inserted into a collection tube, and the setup was mixed using a vortex. The sample was 120 pipetted into the upper buffer reservoir of the filter tube. The whole high-purity filter tube assembly was 121 placed in a standard table-top centrifuge and centrifuged at × 8,000 g for 1 min. The filter tube was then 122 removed and the rest of the setup was discarded; keeping the collection tube containing the filtrate 123

Subsequently, 500 μl inhibitor removal buffer was added to the supernatant and was centrifuged for one 124 min at 8,000 × g. Finally, the supernatant was removed from the collection tube, 500 μl wash buffer was 125 added to it, and centrifuged for 1 min at 8,000 × g. 126

The flow-through was scrapped, and the whole high purity assembly was centrifuged at full speed for 127 another 30 sec. The elution buffer was added, and the DNA was precipitated in 100 μl TE. Brief 128 centrifugation (15,000 g for 1 min) was used to separate the cell debris, and 1 μl of the supernatant was 129 used for the PCR. The extracted DNA was stored at -20 ºC. 130

We used the PCR to detect Candida spp. The PCR reaction was run in a cumulative volume of 25 μl, 132 containing 1 μl of each of reverse and forward primers, 2 μl of prototype DNA, 12.5 μl of master mix 133 (Amplicon, Denmark), and water until it reached the final volume. The amplification was done using the 134 internal transcribed spacers 1 (ITS1) and ITS4 primers based on the following protocol: 10 min of primary denaturation at 95 ºC, 40 cycles of denaturation for 20 sec at 95 ºC, annealing for 20 sec at 62 ºC, an 136 expansion for 20 sec at 72 ºC, and a final extension for 5 min at 72 ºC. Eventually, the products were run 137 on a 2% agarose gel. The HWP1 gene amplification using the paired primers HWP1-F (5′-138 GCTACCACTTCAGAATCATCATC-3′) and HWP1-R (5′-GCACCTTCAGTCGTAGAGACG-3′) was 139 done as described previously for Candida albicans complex [14, 22] . 140

The Clinical and Laboratory Standards Institute (CLSI) M60 approach was used to assess the minimum 142 

Statistical analysis was carried out using SPSS software (version 16.0). Descriptive test was performed to 151 describe the demographic characteristics, and chi-square test was performed to demonstrate any statistically 152 significant relationship between the variables explored in this study. The MICs range and MICs 90 of all 153 antifungals were calculated. 154

Candida colonization was confirmed in 69 (69%) of the 100 COVID-19 patients under MV. Of these, 37/69 156 (53.6%) patients were males; the mean age of all patients at presentation was 61.1 years (range = 21-88 157 years). Based on the PCR sequencing results, C. albicans (55; 79.7%) was the most common spp. followed 158 by, C. glabrata (12; 17.4%). The co-infection of C. albicans and C. glabrata was seen in two cases (2.9%). 159

In this research, two (2.9%) Candida africana were detected by the HWP1 gene amplification (Figure 1 ), 160 and no Candida dubliniensis was found. 161

On the first day of admission, D0, all 69 patients using MV had Candida spp. airway colonization, while 162 there was no substantial difference in the cause for ICU entry (P>0.05). Moreover, at D0, C. albicans was 163 responsible for 79.7 % of Candida respiratory tract colonization. In 63.77% of patients, Candida respiratory 164 colonization had no impact on the severity of COVID-19 (P>0.05) between D0 and D14. In comparison to 165 C. albicans strains, Candida respiratory tract colonization with C. glabrata had influenced the severity of 166 COVID-19 in critically ill patients following adjustment for the risk factors of COVID-19 (P<0.05). 167

The most common underlying diseases among patients with Candida colonization included diabetes (28 168 cases), malignancy (8 cases), kidney disorders (11 cases), cardiovascular diseases (7 cases), and one 1 case 169 each of pregnancy and hyperthyroidism. Whereas patients with Candida colonization had diabetes (40.6%) 170 and kidney disorders (16%) as their main underlying diseases, headache (97.1%), fever (85.5%), myalgia 171 (91.6%), arthralgia (49.3%), gastrointestinal symptoms (71%), and dyspnea (100%) were most frequent 172 symptoms at presentation depending on patients' status of Candida colonization. Table 1  173 The clinical course and disease outcome of patients with and without Candida colonization is being been 174 demonstrated in Table 2 . 175 Table 3 . Characteristics of patients, clinical findings, signs and symptoms, laboratory findings, and 176 outcome in patients colonized with C. albicans, patients colonized with C. glabrata and non-colonized 177 patients 178 

Although microbial colonization is an important factor in the development of secondary infections, Candida 189 pneumonia-as a secondary infection following airways colonization -is seldom reported even in the 190 intensive care unit (ICU). Thus, the common consensus is that anti-Candida therapy is rarely necessary in 191 most cases and it should be managed as airways colonization in which Candida spp. are being isolated [24] . Various degrees of resistance to voriconazole, itraconazole and fluconazole were seen in C. albicans and 245 C. glabrata strains. Antifungal agents showed 100% sensitivity against the two C. africana strains. In 246 another study, 100%, 30%, and 40% of the Candida auris isolates were resistant to FCZ, combination of 247 FCZ and voriconazole, and combination of FCZ and AMB, respectively, and only one Candida glabrata 248 isolate was resistant against echinocandin [8, [36] [37] [38] [39] [40] [41] [42] . 249

In this study, the use of automatic ventilation, respiratory colonization, or infection with Candida spp. was 251 not recognized to influence variables of the infection or death caused by COVID-19. Although "Candida 252 pneumonia" is rarely being reported in critically ill patients, Candida airway colonization mainly by 253 J o u r n a l P r e -p r o o f Candida albicans is common especially among patients with diabetes, malignancies, and kidney disorders. 254

In this study, amphotericin B and caspofungin showed superior activity against all Candida spp. 

The authors declare that there is no conflict of interest. 

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Age, yr, median (range)

Blood group, A, n (%)

Age, yr, median (range)

FBS, mg/dl, median (range)

BUN, mg/dl, median (range)

CRP, mg/dl, median (range) 96.1 (2-382) 109 (21-344) 85